# Patent

## Title
Albumin fusion proteins

## Abstract

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disordrs or conditions using albumin fusion proteins of the invention.

## Background
This application claims the benefit of priority under 35 U.S.C. § 119(e) based on the following U.S. provisional applications: No. 60/229,358 filed on Apr. 12, 2000; No. 60/199,384 filed on Apr. 25, 2000; and No. 60/256,931 filed on Dec. 21, 2000. Each of the provisional applications is hereby incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION
The invention relates generally to Therapeutic proteins (including, but not limited to, a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or fragments or variants of albumin. The invention further relates to Therapeutic proteins (including, but not limited to, a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or fragments or variants of albumin, that exhibit extended shelf-life and/or extended or therapeutic activity in solution. These fusion proteins are herein collectively referred to as “albumin fusion proteins of the invention.” The invention encompasses therapeutic albumin fusion proteins, compositions, pharmaceutical compositions, formulations and kits. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention using these nucleic acids, vectors, and/or host cells.
The invention is also directed to methods of in vitro stabilizing a Therapeutic protein via fusion or conjugation of the Therapeutic protein to albumin or fragments or variants of albumin.
Human serum albumin (HSA, or HA), a protein of 585 amino acids in its mature form (as shown in FIG. 15 or in SEQ ID NO:18), is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. At present, HA for clinical use is produced by extraction from human blood. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991.
The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a carrier and transporter of polypeptides in vivo. The use of albumin as a component of an albumin fusion protein as a carrier for various proteins has been suggested in WO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminal fragments of HA for fusions to polypeptides has also been proposed (EP 399 666). Fusion of albumin to the Therapeutic protein may be achieved by genetic manipulation, such that the DNA coding for HA, or a fragment thereof, is joined to the DNA coding for the Therapeutic protein. A suitable host is then transformed or transfected with the fused nucleotide sequences, so arranged on a suitable plasmid as to express a fusion polypeptide. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo e.g. from a transgenic organism.
Therapeutic proteins in their native state or when recombinantly produced, such as interferons and growth hormones, are typically labile molecules exhibiting short shelf-lives, particularly when formulated in aqueous solutions. The instability in these molecules when formulated for administration dictates that many of the molecules must be lyophilized and refrigerated at all times during storage, thereby rendering the molecules difficult to transport and/or store. Storage problems are particularly acute when pharmaceutical formulations must be stored and dispensed outside of the hospital environment. Many protein and peptide drugs also require the addition of high concentrations of other protein such as albumin to reduce or prevent loss of protein due to binding to the container. This is a major concern with respect to proteins such as IFN. For this reason, many Therapeutic proteins are formulated in combination with large proportion of albumin carrier molecule (100-1000 fold excess), though this is an undesirable and expensive feature of the formulation.
Few practical solutions to the storage problems of labile protein molecules have been proposed. Accordingly, there is a need for stabilized, long lasting formulations of proteinaceous therapeutic molecules that are easily dispensed, preferably with a simple formulation requiring minimal post-storage manipulation.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery that Therapeutic proteins may be stabilized to extend the shelf-life, and/or to retain the Therapeutic protein's activity for extended periods of time in solution, in vitro and/or in vivo, by genetically or chemically fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to stabilize the protein and/or its activity. In addition it has been determined that the use of albumin-fusion proteins or albumin conjugated proteins may reduce the need to formulate protein solutions with large excesses of carrier proteins (such as albumin, unfused) to prevent loss of Therapeutic proteins due to factors such as binding to the container.
The present invention encompasses albumin fusion proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or a fragment (portion) or variant of albumin. The present invention also encompasses albumin fusion proteins comprising a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) fused to albumin or a fragment (portion) or variant of albumin, that is sufficient to prolong the shelf life of the Therapeutic protein, and/or stabilize the Therapeutic protein and/or its activity in solution (or in a pharmaceutical composition) in vitro and/or in vivo. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells.
The invention also encompasses pharmaceutical formulations comprising an albumin fusion protein of the invention and a pharmaceutically acceptable diluent or carrier. Such formulations may be in a kit or container. Such kit or container may be packaged with instructions pertaining to the extended shelf life of the Therapeutic protein. Such formulations may be used in methods of treating, preventing, ameliorating, or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising the step of administering the pharmaceutical formulation to the patient.
In other embodiments, the present invention encompasses methods of preventing treating, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indication Y” column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to a Therapeutic protein (or fragment or variant thereof) disclosed in the “Therapeutic Protein X” column of Table 1 (in the same row as the disease or disorder to be treated is listed in the “Preferred Indication Y” column of Table 1) in an amount effective to treat prevent or ameliorate the disease or disorder.
In another embodiment, the invention includes a method of extending the shelf life of a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) comprising the step of fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to extend the shelf-life of the Therapeutic protein. In a preferred embodiment, the Therapeutic protein used according to this method is fused to the albumin, or the fragment or variant of albumin. In a most preferred embodiment, the Therapeutic protein used according to this method is fused to albumin, or a fragment or variant of albumin, via recombinant DNA technology or genetic engineering.
In another embodiment, the invention includes a method of stabilizing a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) in solution, comprising the step of fusing or conjugating the Therapeutic protein to albumin or a fragment (portion) or variant of albumin, that is sufficient to stabilize the Therapeutic protein. In a preferred embodiment, the Therapeutic protein used according to this method is fused to the albumin, or the fragment or variant of albumin. In a most preferred embodiment, the Therapeutic protein used according to this method is fused to albumin, or a fragment or variant of albumin, via recombinant DNA technology or genetic engineering.
The present invention further includes transgenic organisms modified to contain the nucleic acid molecules of the invention, preferably modified to express the albumin fusion proteins encoded by the nucleic acid molecules.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts the extended shelf-life of an HA fusion protein in terms of the biological activity (Nb2 cell proliferation) of HA-hGH remaining after incubation in cell culture media for up to 5 weeks at 37° C. Under these conditions, hGH has no observed activity by week 2.
FIG. 2 depicts the extended shelf-life of an HA fusion protein in terms of the stable biological activity (Nb2 cell proliferation) of HA-hGH remaining after incubation in cell culture media for up to 3 weeks at 4, 37, or 50° C. Data is normalized to the biological activity of hGH at time zero.
FIGS. 3A and 3B compare the biological activity of HA-hGH with hGH in the Nb2 cell proliferation assay. FIG. 3A shows proliferation after 24 hours of incubation with various concentrations of hGH or the albumin fusion protein, and FIG. 3B shows proliferation after 48 hours of incubation with various concentrations of hGH or the albumin fusion protein.
FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as the base vector into which polynucleotides encoding the Therapeutic proteins (including polypeptide and fragments and variants thereof) may be cloned to form HA-fusions. Plasmid Map key:
PRB1p: PRB1 S. cerevisiae promoter; FL: Fusion leader sequence; rHA: cDNA encoding HA: ADH1t: ADH1 S. cerevisiae terminator; T3: T3 sequencing primer site; T7: T7 sequencing primer site; Amp R: β-lactamase gene; ori: origin of replication. Please note that in the provisional applications to which this application claims priority, the plasmid in FIG. 4 was labeled pPPC0006, instead of pPPC0005. In addition the drawing of this plasmid did not show certain pertinent restriction sites in this vector. Thus in the present application, the drawing is labeled pPPC0005 and more restriction sites of the same vector are shown.
FIG. 5 compares the recovery of vial-stored HA-IFN solutions of various concentrations with a stock solution after 48 or 72 hours of storage.
FIG. 6 compares the activity of an HA-α-IFN fusion protein after administration to monkeys via IV or SC.
FIG. 7 describes the bioavailability and stability of an HA-α-IFN fusion protein.
FIG. 8 is a map of an expression vector for the production of HA-_-IFN.
FIG. 9 shows the location of loops in HA.
FIG. 10 is an example of the modification of an HA loop.
FIG. 11 is a representation of the HA loops.
FIG. 12 shows the HA loop IV.
FIG. 13 shows the tertiary structure of HA.
FIG. 14 shows an example of a scFv-HA fusion
FIG. 15 shows the amino acid sequence of the mature form of human albumin (SEQ ID NO: 18) and a polynucleotide encoding it (SEQ ID NO: 17).



DETAILED DESCRIPTION
As described above, the present invention is based, in part, on the discovery that a Therapeutic protein (e.g., a polypeptide, antibody, or peptide, or fragments and variants thereof) may be stabilized to extend the shelf-life and/or retain the Therapeutic protein's activity for extended periods of time in solution (or in a pharmaceutical composition) in vitro and/or in vivo, by genetically fusing or chemically conjugating the Therapeutic protein, polypeptide or peptide to all or a portion of albumin sufficient to stabilize the protein and its activity.
The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases or disorders. As used herein, “albumin fusion protein” refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or chemical conjugation to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may be referred to as a “portion”, “region” or “moiety” of the albumin fusion protein (e.g., a “Therapeutic protein portion” or an “albumin protein portion”).
In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein (e.g., as described in Table 1) and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.
In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein. In a further preferred embodiment, the Therapeutic protein portion of the albumin fusion protein is the extracellular soluble domain of the Therapeutic protein. In an alternative embodiment, the Therapeutic protein portion of the albumin fusion protein is the active form of the Therapeutic protien.
In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin.
Therapeutic Proteins
As stated above, an albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion or chemical conjugation.
As used herein, “Therapeutic protein” refers to proteins, polypeptides, antibodies, peptides or fragments or variants thereof, having one or more therapeutic and/or biological activities. Therapeutic proteins encompassed by the invention include but are not limited to, proteins, polypeptides, peptides, antibodies, and biologics. (The terms peptides, proteins, and polypeptides are used interchangeably herein.) It is specifically contemplated that the term, “Therapeutic protein” encompasses antibodies and fragments and variants thereof. Thus an albumin fusion protein of the invention may contain at least a fragment or variant of a Therapeutic protein, and/or at least a fragment or variant of an antibody. Additionally, the term “Therapeutic protein” may refer to the endogenous or naturally occurring correlate of a Therapeutic protein.
By a polypeptide displaying a “therapeutic activity” or a protein that is “therapeutically active” is meant a polypeptide that possesses one or more known biological and/or therapeutic activities associated with a Therapeutic protein such as one or more of the Therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a “Therapeutic protein” is a protein that is useful to treat, prevent or ameliorate a disease, condition or disorder. As a non-limiting example, a “Therapeutic protein” may be one that binds specifically to a particular cell type (normal (e.g., lymphocytes) or abnormal e.g., (cancer cells)) and therefore may be used to target a compound (drug, or cytotoxic agent) to that cell type specifically.
In another non-limiting example, a “Therapeutic protein” is a protein that has a biological activity, and in particular, a biological activity that is useful for treating preventing or ameliorating a disease. A non-inclusive list of biological activities that may be possessed by a Therapeutic protein includes, enhancing the immune response, promoting angiogenesis, inhibiting angiogenesis, regulating hematopoietic functions, stimulating nerve growth, enhancing an immune response, inhibiting an immune response, or any one or more of the biological activities described in the “Biological Activities” section below.
As used herein, “therapeutic activity” or “activity” may refer to an activity whose effect is consistent with a desirable therapeutic outcome in humans, or to desired effects in non-human mammals or in other species or organisms. Therapeutic activity may be measured in vivo or in vitro. For example, a desirable effect may be assayed in cell culture. As an example, when hGH is the Therapeutic protein, the effects of hGH on cell proliferation as described in Example 1 may be used as the endpoint for which therapeutic activity is measured. Such in vitro or cell culture assays arm commonly available for many Therapeutic proteins as described in the art. Examples of assays include, but are not limited to those described herein in the Examples section or in the “Exemplary Activity Assay” column of Table 1.
Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, such as cell surface and secretory proteins, are often modified by the attachment of one or more oligosaccharide groups. The modification, referred to as glycosylation, can dramatically affect the physical properties of proteins and can be important in protein stability, secretion, and localization. Glycosylation occurs at specific locations along the polypeptide backbone. There are usually two major types of glycosylation: glycosylation characterized by O-linked oligosaccharides, which are attached to serine or threonine residues; and glycosylation characterized by N-linked oligosaccharides, which are attached to asparagine residues in an Asn-X-Ser/Thr sequence, where X can be any amino acid except proline. N-acetylneuramic acid (also known as sialic acid) is usually the terminal residue of both N-linked and O-linked oligosaccharides. Variables such as protein structure and cell type influence the number and nature of the carbohydrate units within the chains at different glycosylation sites. Glycosylation isomers are also common at the same site within a given cell type.
For example, several types of human interferon are glycosylated. Natural human interferon-α2 is O-glycosylated at threonine 106, and N-glycosylation occurs at asparagine 72 in interferon-α14 (Adolf et al., J. Biochem 276:511 (1991); Nyman T A et al., J. Biochem 329:295 (1998)). The oligosaccharides at asparagine 80 in natural interferon-β1α may play an important factor in the solubility and stability of the protein, but may not be essential for its biological activity. This permits the production of an unglycosylated analog (interferon-β1b) engineered with sequence modifications to enhance stability (Hosoi et al., J. Interferon Res. 8:375 (1988; Karpusas et al., Cell Mol Life Sci 54:1203 (1998); Knight, J. Interferon Res. 2:421 (1982); Runkel et al., Pharm Res 15:641 (1998); Lin, Dev. Biol. Stand. 96:97 (1998))1. Interferon-y contains two N-linked oligosaccharide chains at positions 25 and 97, both important for the efficient formation of the bioactive recombinant protein, and having an influence on the pharmacokinetic properties of the protein (Sareneva et al., Eur. J. Biochem 242:191 (1996); Sareneva et al., Biochem J. 303:831 (1994); Sareneva et al., J. Interferon Res. 13:267 (1993)). Mixed O-linked and N-linked glycosylation also occurs, for example in human erythropoietin, N-linked glycosylation occurs at asparagine residues located at positions 24, 38 and 83 while O-linked glycosylation occurs at a serine residue located at position 126 (Lai et al., J. Biol. Chem. 261:3116 (1986); Broudy et al., Arch. Biochem. Biophys. 265:329 (1988)).
Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, as well as analogs and variants thereof, may be modified so that glycosylation at one or more sites is altered as a result of manipulation(s) of their nucleic acid sequence, by the host cell in which they are expressed, or due to other conditions of their expression. For example, glycosylation isomers may be produced by abolishing or introducing glycosylation sites, e.g., by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or unglycosylated recombinant proteins, may be produced by expressing the proteins in host cells that will not glycosylate them, e.g. in E. coli or glycosylation-deficient yeast. These approaches are described in more detail below and are known in the art.
Therapeutic proteins (particularly those disclosed in Table 1) and their nucleic acid sequences are well known in the art and available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, and GenSeq as shown in Table 1.
Additional Therapeutic proteins corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, one or more of the Therapeutic proteins or peptides disclosed in the “Therapeutic Protein X” column of Table 1, or fragment or variable thereof.
Table 1 provides a non-exhaustive list of Therapeutic proteins that correspond to a Therapeutic protein portion of an albumin fusion protein of the invention. The “Therapeutic Protein X” column discloses Therapeutic protein molecules followed by parentheses containing scientific and brand names that comprise, or alternatively consist of, that Therapeutic protein molecule or a fragment or variant thereof. “Therapeutic protein X” as used herein may refer either to an individual Therapeutic protein molecule (as defined by the amino acid sequence obtainable from the CAS and Genbank accession numbers), or to the entire group of Therapeutic proteins associated with a given Therapeutic protein molecule disclosed in this column. The “Exemplary Identifier” column provides Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or Genbank Accession Numbers ((e.g., Locus ID, NP_XXXXX (Reference Sequence Protein), and XP_XXXXX (Model Protein) identifiers available through the national Center for Biotechnology Information (NCBI) webpage at www.ncbi.nlm.nih.gov) that correspond to entries in the CAS Registry or Genbank database which contain an amino acid sequence of the Therapeutic Protein Molecule or of a fragment or variant of the Therapeutic Protein Molecule. In addition GenSeq Accession numbers and/or journal publication citations are given to identify the exemplary amino acid sequence for some polypeptides. The summary pages associated with each of these CAS and Genbank and GenSeq Accession Numbers as well as the cited journal publications (e.g., PubMed ID number (PMID)) are each incorporated by reference in their entireties, particularly with respect to the amino acid sequences described therein. The “PCT/Patent Reference” column provides U.S. Patent numbers, or PCT International Publication Numbers corresponding to patents and/or published patent applications that describe the Therapeutic protein molecule. Each of the patents and/or published patent applications cited in the “PCT/Patent Reference” column are herein incorporated by reference in their entreties. In particular, the amino acid sequences of the specified polypeptide set forth in the sequence listing of each cited “PCT/Patent Reference”, the variants of these amino acid sequences (mutations, fragments, etc.) set forth, for example, in the detailed description of each cited “PCT/Patent Reference”, the therapeutic indications set forth, for example, in the detailed description of each cited “PCT/Patent Reference”, and the activity assay for the specified polypeptide set forth in the detailed description, and more particularly, the examples of each cited “PCT/Patent Reference” are incorporated herein by reference. The “Biological activity” column describes Biological activities associated with the Therapeutic protein molecule. The “Exemplary Activity Assay” column provides references that describe assays which may be used to test the therapeutic and/or biological activity of a Therapeutic protein or an albumin fusion protein of the invention comprising a Therapeutic protein X portion. Each of the references cited in the “Exemplary Activity Assay” column are herein incorporated by reference in their entireties, particularly with respect to the description of the respective activity assay described in the reference (see Methods section, for example) for assaying the corresponding biological activity set forth in the “Biological Activity” column of Table 1. The “Preferred Indication Y” column describes disease, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by Therapeutic protein X or an albumin fusion protein of the invention comprising a Therapeutic protein X portion.















Therapeutic
Exemplary






Protein X
Identifier
PCT/Patent Number
Biological Activity
Exemplary Activity Assay
Preferred Indication Y







BMP-1
GeneSeq
WO8800205
BMP1 belongs to the
BMP-1 activity can be determined
Induction of Cartilage, Tissue



Accession P80618

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic proteins induce
Jan; 27(1): 84-8; Eur J Biochem 1996





cartilage and bone formation,
Apr 1; 237(1): 295-302; J Biol Chem,





play an important role in
Vol. 274, Issue 16, 10897-10902,





nephrogenesis, and play an
April 16, 1999; and Hogan, B. L. M.





important role in the
(1996) Genes Dev. 10, 1580-1594.





development of many organs,





including lung, heart, teeth, gut,





skin, and particularly the kidney.


BMP-2
GeneSeq
WO8800205
BMP-2 belongs to the
BMP-2 activity can be determined
Induction of Cartilage, Tissue



Accession P80619

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-2B
GeneSeq
U.S. Pat. No. 5631142
BMP-2b belongs to the
BMP-2b activity can be determined
Induction of Cartilage, Tissue



Accession

transforming growth factor-beta
using the following assays known in
and Bone Growth, and



W24850

(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-4
GeneSeq
WO0020591
BMP-4 belongs to the
BMP-4 activity can be determined
Induction of Cartilage, Tissue



Accession B02796

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-5
GeneSeq
WO0020591
BMP-5 belongs to the
BMP-5 activity can be determined
Induction of Cartilage, Tissue



Accession B02797

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-6
GeneSeq
U.S. Pat. No. 5187076
BMP-6 belongs to the
BMP-6 activity can be determined
Induction of Cartilage, Tissue



Accession R32904

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


Osteogenic
GeneSeq
WO9734626
OP-1 belongs to the transforming
OP-1 activity can be determined
Induction of Cartilage, Tissue


Protein-1; OP-1;
Accession

growth factor-beta (TGFB)
using the following assays known in
and Bone Growth, and


BMP-7
W34783

superfamily. Bone morphogenic
the art: Nat Genet. 2001
Diabetes





protein induces bone formation.
Jan; 27(1): 84-8; Eur J Biochem 1996






Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


Osteogenic
GeneSeq
WO9406399
OP-2 belongs to the transforming
OP-2 activity can be determined
Induction of Cartilage, Tissue


Protein-2
Accession R57973

growth factor-beta (TGFB)
using the following assays known in
and Bone Growth, and





superfamily. Bone morphogenic
the art: Nat Genet. 2001
Diabetes





protein induces bone formation.
Jan; 27(1): 84-8; Eur J Biochem 1996






Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594


GDF-1
GeneSeq
WO9406449
Members of the TGF-beta family
The effect of GDF-1 on signaling
Developmental disorders,



Accession R60961

of proteins initiate cell signaling
can be assayed by treating Primary
Induction of Cartilage, Tissue





by binding to heteromeric
BAECs transfected with a construct
and Bone Growth, and





receptor complexes of type I
called p3TP-Lux, containing a TGF-
Diabetes





(TbetaRI) and type II (ThetaRII)
beta responsive promoter fused to a





serine/threonine kinase receptors
reporter gene, and measuring





(reviewed by Massague, J. et al.
luciferase gene expression (Wrana





(1994) Trends Cell Biol. 4: 172-
et al., 1994, Nature 370: 341-347).





178; Miyazono, K. et al. (1994)





Adv. Immunol. 55: 181-220).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


BMP-9
GeneSeq
WO9533830
BMP-9 belongs to the
BMP-9 activity can be determined
Induction of Cartilage, Tissue



Accession R86903

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999: and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-10
GeneSeq
WO9426893
BMP-10 belongs to the
BMP-10 activity can be determined
Induction of Cartilage, Tissue



Accession R66202

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-12
GeneSeq
WO9516035
BMP-12 belongs to the
BMP-12 activity can be determined
Induction of Cartilage, Tissue



Accession R78734

transforming growth factor-beta
using the following assays known in
and Bone Growth, and





(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-15
GeneSeq
WO9636710
BMF-15 belongs to the
BMP-15 activity can be determined
Induction of Cartilage, Tissue



Accession

transforming growth factor-beta
using the following assays known in
and Bone Growth, and



W11261

(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-17
GeneSeq
WO9929718
BMP-17 belongs to the
BMP-17 activity can be determined
Induction of Cartilage, Tissue



Accession

transforming growth factor-beta
using the following assays known in
and Bone Growth, and



Y17870

(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP-18
GeneSeq
WO9929718
BMP-18 belongs to the
BMP-18 activity can be determined
Induction of Cartilage, Tissue



Accession

transforming growth factor-beta
using the following assays known in
and Bone Growth, and



Y 17871

(TGFB) superfamily. Bone
the art: Nat Genet. 2001
Diabetes





morphogenic protein induces
Jan; 27(1): 84-8; Eur J Biochem 1996





bone formation.
Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


Inhibin alpha
GeneSeq
WO0020591
The inhibin beta A subunit joins
Tumor suppressor activity of inhibin
Tumor suppression.



Accession B02806

the alpha subunit to form a
can be determined using assays





pituitary FSH secretion inhibitor.
known in the art: Matzuk et al.,





Inhibin has been shown to
Nature 1992 Nov 26; 360(6402): 313-





regulate gonadal stromal cell
9.





proliferation negatively and to





have tumour-suppressor activity.





In addition, serum levels of





inhibin have been shown to





reflect the size of granulosa-cell





tumors and can therefore be used





as a marker for primary as well





as recurrent disease.


Inhibin beta
GeneSeq
WO0020591
The inhibin beta A subunit joins
Tumor suppressor activity of inhibin
Tumor suppression.



Accession B02808

the alpha subunit to form a
can be determined using assays





pituitary FSH secretion inhibitor.
known in the art: Matzuk et al.,





Inhibin has been shown to
Nature 1992 Nov 26; 360(6402): 313-





regulate gonadal stromal cell
9.





proliferation negatively and to





have tumour-suppressor activity.





In addition, serum levels of





inhibin have been shown to





reflect the size of granulosa-cell





tumors and can therefore be used





as a marker for primary as well





as recurrent disease.


Cerebus Protein
GeneSeq
WO9849296
Cerebus is believed to be
BMP activity, in the presence of the
BMP Antagonist useful for



Accession

involved in the inhibition of
antagonist Cerebus, can be
Osteosarcoma, abnormal bone



W86032

BMP activity
determined using the following
growth






assays known in the art: Nat Genet.






2001 Jan; 27(1): 84-8; Eur J Biochem






1996 Apr 1; 237(1): 295-302; J Biol






Chem, Vol. 274, Issue 16, 10897-






10902, April 16, 1999; and Hogan,






B. L. M. (1996) Genes Dev. 10,






1580-1594.


Soluble BMP
GeneSeq
WO9614579
Soluble BMP receptor kinase
BMP activity, in the presence of the
BMP Antagonist useful for


Receptor Kinase
Accession R95227

protein-3 is involved in the
soluble antagonist BMP receptor
Osteosarcoma, abnormal bone


Protein-3


binding of BMPs. Soluble BMP
kinase protein-3, can be determined
growth





receptor kinase protein-3 is
using the following assays known in





useful as an antagonist for the
the art: Nat Genet. 2001





inhibition of BMP activity
Jan; 27(1): 84-8; Eur J Biochem 1996






Apr 1; 237(1): 295-302; J Biol Chem,






Vol. 274, Issue 16, 10897-10902,






April 16, 1999; and Hogan, B. L. M.






(1996) Genes Dev. 10, 1580-1594.


BMP
GeneSeq
WO9741250
BMPs belong to the transforming
BMP activity, in the presence of the
Bone formation or


Processing
Accession

growth factor-beta (TGFB)
Furin, can be determined using the
Regeneration Abnormalities


Enzyme Furin
W36099

superfamily. Bone morphogenic
following assays known in the art: 





protein induces bone formation.
Nat Genet. 2001 Jan; 27(1): 84-8; Eur






J Biochem 1996 Apr 1; 237(1): 295-






302; J Biol Chem, Vol. 274, Issue






16, 10897-10902, April 16, 1999;






and Hogan, B. L. M. (1996) Genes






Dev. 10, 1580-1594.


TGF-beta 1
GeneSeq
WO9216228
Members of the TGF-beta family
The effect of TGF betas on
useful for treating cancer and



Accession R29657

of proteins initiate cell signaling
signaling can be assayed by treating
to promote wound





by binding to heteromeric
Primary BAECs transfected with a
healing





receptor complexes of type I
construct called p3TP-Lux,





(TbetaRI) and type II (TbetaRII)
containing a TGF-beta responsive





serine/threonine kinase receptors
promoter fused to a reporter gene,





(reviewed by Massague, J. et al.
and measuring luciferase gene





(1994) Trends Cell Biol. 4: 172-
expression (Wrana et al., 1994,





178; Miyazono, K. et al. (1994)
Nature 370: 341-347).





Adv. Immunol. 55: 181-220).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


TGF-beta 2
GeneSeq
EP542679
Members of the TGF-beta family
The effect of TGF betas on
useful for treating cancer and



Accession R39659

of proteins initiate cell signaling
signaling can be assayed by treating
to promote wound





by binding to heteromeric
Primary BAECs transfected with a
healing





receptor complexes of type I
construct called p3TP-Lux,





(TbetaRI) and type II (TbetaRII)
containing a TGF-beta responsive





serine/threonine kinase receptors
promoter fused to a reporter gene,





(reviewed by Massague, J. et al.
and measuring luciferase gene





(1994) Trends Cell Biol. 4: 172-
expression (Wrana et al., 1994,





178; Miyazono, K. et al. (1994)
Nature 370: 341-347).





Adv. Immunol. 55: 181-220).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


ZTGF-beta 9
GeneSeq
WO0015798
Members of the TGF-beta family
The effect of TGF betas on
useful for treating cancer and



Accession

of proteins initiate cell signaling
signaling can be assayed by treating
to promote wound



Y70654

by binding to heteromeric
Primary BAECs transfected with a
healing





receptor complexes of type I
construct called p3TP-Lux,





(TbetaRI) and type II (TbetaRII)
containing a TGF-beta responsive





serine/threonine kinase receptors
promoter fused to a reporter gene,





(reviewed by Massague, J. et al.
and measuring luciferase gene





(1994) Trends Cell Biol. 4: 172-
expression (Wrana et al., 1994,





178; Miyazono, K. et al. (1994)
Nature 370: 341-347).





Adv. Immunol. 55: 181-220).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


Anti-TGF beta

GB2305921
Members of the TGF-beta family
The effect of TGF betas on
Useful for control of fibrosis,


family


of proteins initiate cell signaling
signaling, in the presence of an anti-
immune, and inflammatory


antibodies


by binding to heteromeric
TGF beta antibody, can be assayed
disease





receptor complexes of type I
by treating Primary BAECs





(TbetaRI) and type II (TbetaRII)
transfected with a construct called





serine/threonine kinase receptors
p3TP-Lux, containing a TGF-beta





(reviewed by Massague, J. et al.
responsive promoter fused to a





(1994) Trends Cell Biol. 4: 172-
reporter gene, and measuring





178; Miyazono, K. et al. (1994)
luciferase gene expression (Wrana





Adv. Immunol. 55: 181-220).
et al., 1994, Nature 370: 341-347).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


Latent TGF-
GeneSeq
WO0012551
Members of the TGF-beta family
The effect of TGF betas on
Useful for inhibiting tissue or


beta binding
Accession

of proteins initiate cell signaling
signaling, in the presence of a TGF-
tumor growth


protein II
Y70552

by binding to heteromeric
beta binding protein, can be assayed





receptor complexes of type I
by treating Primary BAECs





(TbetaRI) and type II (TbetaRII)
transfected with a construct called





serine/threonine kinase receptors
p3TP-Lux, containinig a TGF-beta





(reviewed by Massague, J. et al.
responsive promoter fused to a





(1994) Trends Cell Biol. 4: 172-
reporter gene, and measuring





178; Miyazono, K. et al. (1994)
luciferase gene expression (Wrana





Adv. Immunol. 55: 181-220).
et al., 1994, Nature 370: 341-347).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


MP52
GeneSeq
WO9741250
Members of the TGF-beta family
The effect of TGF betas on
Bone formation or



Accession

of proteins initiate cell signaling
signaling can be assayed by treating
Regeneration Abnormalities



W36100

by binding to heteromeric
Primary BAECs transfected with a





receptor complexes of type I
construct called p3TP-Lux,





(TbetaRI) and type II (TbetaRII)
containing a TGF-beta responsive





serine/threonine kinase receptors
promoter fused to a reporter gene,





(reviewed by Massague, J. et al.
and measuring luciferase gene





(1994) Trends Cell Biol. 4: 172-
expression (Wrana et al., 1994,





178; Miyazono, K. et al. (1994)
Nature 370: 341-347).





Adv. Immunol. 55: 181-220).





Activation of this heteromeric





receptor complex occurs when





TGF-beta. binds to TbetaRII,





which then recruits and





phosphorylates TbetaRI.





Activated TbetaRI then





propagates the signal to





downstream targets (Chen, F.





and Weinberg, R. A. (1995)





PNAS92: 1565-1569; Wrana, J.





L. et al. (1994) Nature 370: 341-





347).


b57 Protein
GeneSeq
WO9837195
BMPs are involved in the
BMP activity, in the presence of b57
BMP Antagonist useful for



Accession

induction of bone formation.
protein, can be determined using the
Osteosarcoma, abnormal bone



W69293

Specific antagonists are useful is
following assays known in the art: 
growth





preventing this activity from
Nat Genet. 2001 Jan; 27(1): 84-8; Eur





occurring.
J Biochem 1996 Apr 1; 237(1): 295-






302; J Biol Chem, Vol. 274, Issue






16, 10897-10902, April 16, 1999;






and Hogan, B. L. M. (1996) Genes






Dev. 10, 1580-1594.


Resistin
GeneSeq
WO0064920
This gene belongs to the family
Ability of resistin to influence type
Type II diabetes and



Accession

defined by mouse FIZZ1 and
II diabetes can be determined using
Syndrome X



W69293

FIZZ3/Resistin genes. The
assays known in the art: Pontoglio et





characteristic feature of this
al., J Clin Invest 1998 May





family is the C-terminal stretch
15; 101(10): 2215-22.





of 10 cys residues with identical





spacing. The mouse homolog of





this protein is secreted by





adipocytes, and may be the





hormone potentially linking





obesity to type II diabetes.


Galectin-4
GeneSeq
WO9703190
Galectins are a family of
Ability of Galectin-4 polypeptides
Lactose intolerance



Accession

carbohydrate-binding proteins
to bind lactose can be determined



W11841

characterized by an affinity for
using assays known in the art: Wada





beta-galactoside containing
et al., J Biol Chem 1997 Feb





glycoconjugates.
28; 272(9): 6078-86.


APM-1; ACRP-
GeneSeq
WO0026363
ACPR30 gene is exclusively
Ability of ACRP30 polypeptides to
Obesity, Metabolic disorders,


30; Famoxin
Accession

expressed in adipose tissue.
influence obesity and fat oxidation
Lipid Metabolism; Hormone



Y71035

ACRP30 is thought to increase
can be determined using assays
Secretion





fatty acid oxidation by muscle
known in the art: Fruebis et al., Proc





tissue.
Natl Acad Sci USA 2001 Feb






13; 98(4): 2005-10.


ACRP-30
GeneSeq
WO0063376
ACPR30 gene is exclusively
Ability of ACRP30 homologue
Obesity, Metabolic disorders,


Homologue,
Accession B30234

expressed in adipose tissue.
polypeptides to influence obesity
Lipid Metabolism; Hormone


Complement


ACRP30 is thought to increase
and fat oxidation can be determined
Secretion


Component Clq


fatty acid oxidation by muscle
using assays known in the art: 


C


tissue.
Fruebis et al., Proc Natl Acad Sci 






USA 2001 Feb 13; 98(4): 2005-10.


Calpain-10a
GeneSeq
WO0023603
Calpain is believed to play a role
Ability of Calpain-10 to influence
Diabetes mellitus; Regulation



Accession

in insulin secretion and insulin
type II diabetes can be determined
of Insulin secretory response;



Y79567

activity, and therefore may be
using assays known in the art: 
Insulin medicated glucose





useful in the treatment of type II
Pontoglio et al., J Clin Invest 1998
transport disorders





diabetes.
May 15; 101(10): 2215-22.


Calpain-10b
GeneSeq
WO0023603
Calpain is believed to play a role
Ability of Calpain-10 to influence
Diabetes mellitus; Regulation



Accession

in insulin secretion and insulin
type II diabetes can be determined
of Insulin in secretory response;



Y79568

activity, and therefore may be
using assays known in the art: 
Insulin mediated glucose





useful in the treatment of type II
Pontoglio et al., J Clin Invest 1998
transport disorders





diabetes.
May 15; 101(10): 2215-22.


Calpain-10c
GeneSeq
WO0023603
Calpain is believed to play a role
Ability of Calpain-10 to influence
Diabetes mellitus; Regulation



Accession

in insulin secretion and insulin
type II diabetes can be determined
of Insulin secretory response,



Y79569

activity, and therefore may be
using assays known in the art: 
Insulin mediated glucose





useful in the treatment of type II
Pontoglio et al., J Clin Invest 1998
transport disorders





diabetes.
May 15; 101(10): 2215-22.


PDGF-D
GeneSeq
WO0027879
Vascular Endothelial Growth
Proliferation assay using NR6R-
Wound Healing;



Accession

Factor
3T3 cells (Rizzino 1988 Cancer
Atherosclerosis



Y71130


Res. 48: 4266)


FasL
GeneSeq
WO9936079
Activities associated with
Activity can be determined using
Apoptosis-related disorders;



Accession

apoptosis and immune system
Apoptosis assays known in the art: 
Autoimmune disorders; Graft-



Y28594

functions.
Walczak et al. (1996) EMBOJ 16: 
v-Host disorders






5386-5397.


Chondromodulin-
GeneSeq
WO0029579
Chondromodulin proteins are
Ability of Chondromodulin-like
Antiangiogenic agent;


like protein
Accession

cartilage proteins thought to
protein to inhibit vasculatization can
Osteoblast proliferation



Y71262

confer resistance to
be determined using assays known
stimulator; prevents





angiogenesis, and thus are useful
in the art: Hiraki et al., J Biol Chem
vascularization of cartilage





as anti-angiogenic agents that
1997 Dec 19; 272(51): 32419-26.
tissue; Useful to treat cancer.





may have utility in combating





cancer.


Patched
GeneSeq
U.S. Pat. No. 5837538
Patched is a tumour-suppressor
Ability of soluble Patched to bind to
Receptor for Hedgehog



Accession

receptor for Sonic hedgehog
and inhibit the activities of shh can
cellular proliferation signaling



W72969

(shh), which is a protein that
be determined using assays known
molecule. This receptor is





controls developmental
in the art: Stone et al., Nature 1996
useful as a means of





patterning and growth.
Nov 14; 384(6605): 129-34.
preventing cellular







proliferation via the shh







signaling pathway, thus useful







for cancers.


Patched-2
GeneSeq
WO9953058
Patched is a tumour-suppressor
Ability of soluble Patched to bind to
Receptor for Hedgehog



Accession

receptor for Sonic hedgehog
and inhibit the activities of shh can
cellular proliferation signaling



Y43261

(shh), which is a protein that
be determined using assays known
molecule. This receptor is





controls developmental
in the art: Stone et al., Nature 1996
useful as a means of





patterning and growth.
Nov 14; 384(6605): 129-34.
preventing cellular







proliferation via the shh







signaling pathway, thus useful







for cancers.


Maspin;
GeneSeq
WO9405804
Maspin is a member of the serpin
The inhibitory effects of Maspin and
Tumor suppressor which is


Protease
Accession R50938

family of serine protease
other protease inhibitors can be
down-regulated in breast


Inhibitor 5


inhibitors that is thought to
assayed using methods known in the
cancers. The mapsin protein





suppress tumor metastasis.
art such as a labeled protease
has tumour






substrate, for example, Universal
suppressing and invasion






Protease Substrate (casein,
suppressing activity






resorufin-labeled): Roche Molecular






Biochemicals, Cat. No. 1080733.


Endostatin
GeneSeq
WO0064946
Endostatin is believed to inhibit
The inhibitory effects of endostatin
Anti-angiogenic activity.



Accession B28399

effects of capillary endothelial
can be assayed using assays
Useful in the prevention and/or





cell proliferation.
disclosed by Cao et al (1996) J.
treatment of cancers.






Biol. Chem. 271 29461-29467


aFGF; FGF-1
GeneSeq
EP298723
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession P94037


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as






Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


bFGF; FGF2
GeneSeq
FR2642086
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession R06685


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as






Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-3; INT-2
GeneSeq
WO9503831
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession R07824


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as






Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-4; HST-1;
GeneSeq
WO9503831
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and


HBGF-4
Accession R07825


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as






Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-5
GeneSeq
WO9730155
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as



W22600


Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-6; Heparin
GeneSeq
EP613946
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and


binding secreted
Accession R58555


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as


transforming



Res. 48: 4266); Examples 23 and 39
epithelial cells and


factor-2



disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-8
GeneSeq
WO9524928
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession R80783


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as






Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-9; Glia
GeneSeq
WO9503831
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and


activating factor
Accession R70822


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as






Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-12;
GeneSeq
WO9635708
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and


Fibroblast
Accession


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as


growth factor
W06309


Res. 48: 4266); Examples 23 and 39
epithelial cells and


homologous



disclosed herein.
keratinocytes. Antagonists


factor-1




may be useful as anti-cancer







agents


FGF-15
GeneSeq
WO9927100
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as



Y08582


Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-16
GeneSeq
WO9918128
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as



Y05474


Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


FGF-18
GeneSeq
WO9927100
Fibroblast Growth Factor
Proliferation assay using NR6R-
Promotion of growth and



Accession


3T3 cells (Rizzino 1988 Cancer
proliferation of cells, such as



Y08590


Res. 48: 4266); Examples 23 and 39
epithelial cells and






disclosed herein.
keratinocytes. Antagonists







may be useful as anti-cancer







agents


flt-3 ligand
GeneSeq
EP627487
Stem Cell Progenitor
Chemokine activities can be
Promotion of immune cell



Accession R67541


determined using assays known in
growth and/or differentiation






the art: Methods in Molecular






Biology, 2000, vol. 138: Chemokine






Protocols. Edited by: A. E. I.






Proudfoot, T. N. C. Wells, and C. A.






Power. © Humana Press Inc.,






Totowa, NJ


VEGF-110
GeneSeq
WO0013702
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y69417


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-121
GeneSeq
WO0071713
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession B50432

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as






as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-138
GeneSeq
WO9940197
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y43483


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-145
GeneSeq
WO0013702
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y69413


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-162
GeneSeq
WO9940197
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y43484


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-165
GeneSeq
WO0013702
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y69414


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-182
GeneSeq
WO9940197
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y43483


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-189
GeneSeq
WO0013702
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y69415


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-206
GeneSeq
WO0013702
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y69416


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-D
GeneSeq
WO9807832
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and



Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



W53240


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF-E;
GeneSeq
WO9947677
Promotes the growth and/or
VEGF activity can be determined
Promotion of growth and


VEGF-X
Accession

proliferation of endothelial cells
using assays known in the art, such
proliferation of cells, such as



Y33679


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


VEGF
GeneSeq
WO9831794
Receptor for VEGF polypeptides
VEGF activity, in the presence of
VEGF Receptor. Fusion


Receptor; KDR;
Accession


flk-1 polypeptides, can be
protein with the extracellular


flk-1
W69679


determined using assays known in
domain is useful as an anti-






the art, such as those disclosed in
angiogenic agent. Agonists






International Publication No.
may be useful in the






WO0045835, for example.
promotion of angiogenesis


Soluble VEGF
GeneSeq
U.S. Pat. No. 5712380
Receptor for VEGF polypeptides
VEGF activity, in the presence of
VEGF Receptor. Fusion


Receptor
Accession


VEGF Receptor polypeptides, can
protein with the extracellular



W47037


be determined using assays known
domain is useful as an anti-






in the art, such as those disclosed in
angiogenic agent. Agonists






International Publication No.
may be useful in the






WO0045835, for example.
promotion of angiogenesis


flt-1
GeneSeq
WO0021560
Receptor for VEGF polypeptides
VEGF activity, in the presence of
VEGF Receptor. Fusion



Accession


flt-1 polypeptides, can be
protein with the extracellular



Y70751


determined using assays known in
domain is useful as an anti-






the art, such as those disclosed in
angiogenic agent. Agonists






International Publication No.
may be useful in the






WO0045835, for example.
promotion of angiogenesis


VEGF R-3; flt-4
GeneSeq
WO0058511
Receptor for VEGF polypeptides
VEGF activity, in the presence of
VEGFR Receptor. Fusion



Accession B29047


flt-4 polypeptides, can be
protein with the extracellular






determined using assays known in
domain is useful as an anti-






the art, such as those disclosed in
angiogenic agent. Agonists






International Publication No.
may be useful in the






WO0045835, for example.
promotion of angiogenesis


Neuropilin-1
GeneSeq
WO9929858
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and



Accession

Factor
using assays known in the art, such
proliferation of cells, such as



Y06319


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Neuropilin-2
GeneSeq
WO9929858
Vascular Endothelial Growth
VEGF activiiy can be determined
Promotion of growth and



Accession

Factor
using assays known in the art, such
proliferation of cells, such as



Y03618


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Human fast
GeneSeq
WO9730085
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


twitch skeletal
Accession

proteins that are thought to
inhibit angiogenesis can be


muscle troponin
W22597

inhibit angiogenesis. High levels
determined using assays known in


C


may contibute to the difficulty
the art: Proc Natl Acad Sci USA





encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


Human fast
GeneSeq
WO9730085
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


twitch skeletal
Accession

proteins that are thought to
inhibit angiogenesis can be


muscle troponin
W22598

inhibit angiogenesis. High levels
determined using assays known in


I.


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA





encountered in revascularizing
1999 Mar16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


Human fast
GeneSeq
WO9730085
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


twitch skeletal
Accession

proteins that are thought to
inhibit angiogenesis can be


muscle troponin
W22599

inhibit angiogenesis. High levels
determined using assays known in


T


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA





encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


fragment.
GeneSeq
WO9719955
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


myofibrillar
Accession

proteins that are thought to
inhibit angiogenesis can be


protein
W18053

inhibit angiogenesis. High levels
determined using assays known in


Troponin I


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA





encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


myofibrillar
GeneSeq
WO9719955
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


contractile
Accession

proteins that are thought to
inhibit angiogenesis can be


protein
W18054

inhibit angiogenesis. High levels
determined using assays known in


Troponin I


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA





encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


Troponin
GeneSeq
WO9933874
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


peptides
Accessions

proteins that are thought to
inhibit angiogenesis can be



Y29581, Y29582,

inhibit angiogenesis. High levels
determined using assays known in



Y29583, Y29584,

may contibute to the difficulty
the art: . Proc Natl Acad Sci USA



Y29585, and

encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.



Y29586

the ischemic myocardium





after cardiovascular injury.


Human fast
GeneSeq
WO0054770
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


twitch skeletal
Accession B00134

proteins that are thought to
inhibit angiogenesis can be


muscle


inhibit angiogenesis. High levels
determined using assays known in


Troponin


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA


subunit C.


encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


Human fast
GeneSeq
WO0054770
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


twitch skeletal
Accession B00135

proteins that are thought to
inhibit angiogenesis can be


muscle


inhibit angiogenesis. High levels
determined using assays known in


Troponin


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA


subunit I


encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.


Protein


the ischemic myocardium





after cardiovascular injury.


Human fast
GeneSeq
WO0054770
Troponins are contractile
Ability of soluble Troponins to
Anti-angiogenesis


twitch skeletal
Accession B00136

proteins that are thought to
inhibit angiogenesis can be


muscle


inhibit angiogenesis. High levels
determined using assays known in


Troponin


may contibute to the difficulty
the art: . Proc Natl Acad Sci USA


subunit T


encountered in revascularizing
1999 Mar 16; 96(6): 2645-50.





the ischemic myocardium





after cardiovascular injury.


Plasminogen
GeneSeq
WO9013648
PAIs are believed to play a role
Methods that measure plasminogen
Anti-angiogenesis; blood-


Activator
Accession R08411

in cancer, and cardiovascular
activator inhibitor (PAI) aciivity are
clotting disorders.


Inhibitor-I;


disease and blood-clotting
known in the art, for example, assay


PAI-1


disorders.
the ability of PAI to inhibit tissue






plasminogen activator (tPA) or






urokinase (uPA): J Biochem






Biophys Methods 2000 Sep






11; 45(2): 127-40, Breast Cancer Res






Treat 1996; 41(2): 141-6. Methods






that measure anti-angiogenesis






activity are known in the art, for






example, Proc Natl Acad Sci USA






1999 Mar 16; 96(6): 2645-50.


Plasminogen
GeneSeq
DE3722673
PAIs are believed to play a role
Methods that measure plasminogen
Anti-angiogenesis; blood-


Activator
Accession P94160

in cancer, and cardiovascular
activator inhibitor (PAI) activity are
clotting disorders.


Inhibitor-2;


disease and blood-clotting
known in the art, for example, assay


PAI-2


disorders.
the ability of PAI to inhibit tissue






plasminogen activator (tPA) or






urokinase (uPA): J Biochem






Biophys Methods 2000 Sep






11; 45(2): 127-40, Breast Cancer Res






Treat 1996; 41(2): 141-6. Methods






that measure anti-angiogenesis






activity are known in the art, for






example, Proc Natl Acad Sci USA






1999 Mar 16; 96(6): 2645-50.


Plasminogen
GeneSeq
WO9102057
PAIs are believed to play a role
Methods that measure plasminogen
Anti-angiogenesis; blood-


Activator
Accession R10921

in cancer, and cardiovascular
activator inhibitor (PAI) activity are
clotting disorders.


Inhibitor-2;


disease and blood-clotting
known in the art, for example, assay


PAI-2


disorders.
the ability of PAI to inhibit tissue






plasminogen activator (tPA) or






urokinase (uPA): J Biochem






Biophys Methods 2000 Sep






11; 45(2): 127-40, Breast Cancer Res






Treat 1996; 41(2): 141-6. Methods






that measure anti-angiogenesis






activity are known in the art, for






example, Proc Natl Acad Sci USA






1999 Mar 16; 96(6): 2645-50.


Human PAI-1
GeneSeq
WO9105048
PAIs are believed to play a role
Methods that measure plasminogen
Anti-angiogenesis; blood-


mutants
Accessions

in cancer, and cardiovascular
activator inhibitor (PAI) activity are
clotting disorders.



R11755, R11756,

disease and blood-clotting
known in the art, for example, assay



R11757, R11758,

disorders.
the ability of PAI to inhibit tissue



R11759, R11760,


plasminogen activator (tPA) or



R11761, R11762,


urokinase (uPA): J Biochem



and R11763


Biophys Methods 2000 Sep






11; 45(2): 127-40, Breast Cancer Res






Treat 1996; 41(2): 141-6. Methods






that measure anti-angiogenesis






activity are known in the art, for






example, Proc Natl Acad Sci USA






1999 Mar 16; 96(6): 2645-50.


CXCR3; CXC
GeneSeq
WO0018431
Chemokines are a family of
Chemokine activities can be
Soluble CXCR3 polypeptides


chemokine
Accession

related small, secreted proteins
determined using assays known in
may be useful for inhibiting


receptor 3
Y79372

involved in biological processes
the art: Methods in Molecular
chemokine activities and viral





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Modified
GeneSeq
WO9737005
Chemokines are a family of
Chemokine activities can be
Immune disorders


Rantes
Accession

related small, secreted proteins
determined using assays known in



W38129

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


RANTES
GeneSeq
EP905240
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



Y05299

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000. vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


MCP-1a
GeneSeq
WO9509232
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession R73914

related small, secreted proteins
determined using assays known in





involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Yotowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


MCP-1b
GeneSeq
WO9929728
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



Y26176

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Piotocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


MCP-1 receptor
GeneSeq
WO9519436
Chemokines are a family of
Chemokine activities can be
Soluble MCP-1 Receptor



Accession R79165

related small, secreted proteins
determined using assays known in
polypeptides may be useful for





involved in biological processes
the art: Methods in Molecular
inhibiting chemokine activities





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
and viral infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


MCP-3
GeneSeq
WO9509232
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession R73915

related small, secreted proteins
determined using assays known in





involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


MCP-4 receptor
GeneSeq
WO9809171
Chemokines are a family of
Chemokine activities can be
Soluble MCP-4 Receptor



Accession

related small, secreted proteins
determined using assays known in
polypeptides may be useful for



W56689

involved in biological processes
the art: Methods in Molecular
inhibiting chemokine activities





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
and viral infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


RANTES
GeneSeq
U.S. Pat. No. 5652133
Chemokines are a family of
Chemokine activities can be
Soluble RANTES Receptor


receptor
Accession

related small, secreted proteins
determined using assays known in
polypeptides may be useful for



W26588

involved in biological processes
the art: Methods in Molecular
inhibiting chemokine activities





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
and viral infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


CCR5 variant
GeneSeq
WO9854317
Chemokines are a family of
Chemokine activities can be
Soluble CCR5 polypeptides



Accession

related small, secreted proteins
determined using assays known in
may be useful for inhibiting



W88238

involved in biological procceses
the art: Methods in Molecular
chemokine activities and viral





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


CCR7
GeneSeq
U.S. Pat. No. 6153441
Chemokines are a family of
Chemokine activities can be
Soluble CCR7 polypeptides



Accession B50859

related small, secreted proteins
determined using assays known in
may be useful for inhibiting





involved in biological processes
the art: Methods in Molecular
chemokine acitivities and viral





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


CXC3
GeneSeq
WO9727299
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



W23345

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Eotaxin
GeneSeq
WO9700960
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



W10099

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Neurotactin
GeneSeq
U.S. Pat. No. 6013257
Neurotactin may play a role in
Chemotactic leukocyte migration
Immune disorders



Accessions
WO9742224
chemotactic leukocyte migration
assays are known in the art, for



Y77537, W34307,

and brain inflammation
example: J. Immunol. Methods 33,



Y53259, and

processes.
((1980)); Nature 1997 Jun



Y77539


5; 387(6633): 611-7.


Human CKbeta-
GeneSeq
U.S. Pat. No. 6153441
Chemokines are a family of
Chemokine activities can be
Immune disorders


9
Accession B50860

related small, secreted proteins
determined using assays known in





involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Lymphotactin
GeneSeq
WO0073320
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession B50052

related small, secreted proteins
determined using assays known in





involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G


MIP-3 alpha
GeneSeq
WO9801557
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



W44398

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G


MIP-3 beta
GeneSeq
WO9801557
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



W44399

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G


MIP-Gamma
GeneSeq
WO9504158
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession R70798

related small, secreted proteins
determined using assays known in





involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G


Stem Cell
GeneSeq
WO9104274
Chemokines are a family of
Chemokine activities can be
Hematopoietic growth factors


Inhibitory
Accession R11553

related small, secreted proteins
determined using assays known in


Factor


involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000 vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infeciion,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G.


thrombopoietin
GeneSeq
WO9521920
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Hematopoietic growth factors



Accession R79905

the regulation of the growth and
assayed to determine regulation of





differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21 (8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


c-kit ligand;
GeneSeq
EP992579 and
c-kit ligand is thought to 
Chemokine activities can be
Hematopoietic growth factors


SCF; Mast cell
Accession
EP676470
stimulate the proliferation of
determined using assays known in


growth factor;
Y53284, R83978

mast cells, and is able to
the art: Methods in Molecular


MGF;
and R83977.

augment the proliferation of both
Biology, 2000, vol. 138: Chemokine


Fibrosarcoma-


myeloid and lymphoid
Protocols. Edited by: A. E. I.


derived stem


hematopoietic progenitors in
Proudfoot, T. N. C. Wells, and C. A.


cell factor


bone marrow culture. C-kit
Power. © Humana Press Inc.,





ligand is also thought to act
Totowa, NJ





synergistically with other





cytokines.


Platelet derived
GeneSeq
WO0066736
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


growth factor
Accession B48653

Factor
using assays known in the art, such
proliferation of cells, such as






as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Melanoma
GeneSeq
WO9503328
Melanoma inhibiting protein has
Tumor suppressor activity of
Cancer; melenoma


inhibiting
Accession R69811

melanoma-inhibiting activity and
melanoma inhibiting protein can be


protein


can be used to treat cancer
determined using assays known in





(melanoma, glioblastoma,
the art: Matzuk et al., Nature 1992





neuroblastoma, small cell lung
Nov 26; 360(6402): 313-9.





cancer, neuroectodermal





tumors) or as an





immunosuppressant (it inhibits





IL-2 or phytohaemagglutinin





induced proliferation of





peripheral blood lymphocytes.


Glioma-derived
GeneSeq
EP399816
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


growth factor
Accession R08120

Factor
using assays known in the art, such
proliferation of cells, such as






as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Platelet derived
GeneSeq
EP682110
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


growth factor
Accession R84759

Factor
using assays known in the art, such
proliferation of cells, such as


precursor A



as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Platelet derived
GeneSeq
EP682110
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


growth factor
Accession R84760

Factor
using assays known in the art, such
proliferation of cells, such as


precursor B



as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Platetet derived
GeneSeq
EP282317
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


growth factor
Accessions

Factor
using assays known in the art, such
proliferation of cells, such as


Bv-sis
P80595 and


as those disclosed in International
vascular endothelial cells.



P80596


Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Placental
GeneSeq
WO9206194
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


Growth Factor
Accessions

Factor
using assays known in the art, such
proliferation of cells, such as



P23059 and


as those disclosed in International
vascular endothelial cells.



R23060


Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Placental
GeneSeq
DE19748734
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


Growth Factor-
Accession

Factor
using assays known in the art, such
proliferation of cells, such as


2
Y08289


as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Antagonists may be useful as






example.
anti-angiogenic agents, and







may be applicable for cancer


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative1
Accession

the regulation of the growth and
assayed to determine regulation of



Y77244

differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative2
Accession

the regulation of the growth and
assayed to determine regulation of



Y77255

differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 200l Jan; 29(1): 51-8 and






within.


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative3
Accession

the regulation of the growth and
assayed to determine regulation of



Y77262

differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative4
Accession

the regulation of the growth and
assayed to determine regulation of



Y77267

differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative5
Accession

the regulation of the growth and
assayed to determine regulation of



Y77246

differentiaton of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative6
Accession

the regulation of the growth and
assayed to determine regulation of



Y77253

differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


Thrombopoietin
GeneSeq
WO0000612
Thrombopoietin is involved in
Thrombopoietin (TPO) can be
Thrombocytopenia, cancer


derivative7
Accession

the regulation of the growth and
assayed to determine regulation of



Y77256

differentiation of
growth and differentiation of





megakaryocytes and precursors
megakaryocytes. Mol Cell Biol





thereof.
2001 Apr; 21(8): 2659-70; Exp






Hematol 2001 Jan; 29(1): 51-8 and






within.


Fractalkine
GeneSeq
U.S. Pat. No. 6043086
Fractalkine is believed to play a
Fractalkine activity can be
Immune disorders



Accession

role in chemotactic leukocyte
determined using Chemotactic



Y53255

migration and neurological
leukocyte migration assays known





disorders.
in the art, for example: J. Immunol.






Methods 33, ((1980)); Nature 1997






Jun 5; 387(6633): 611-7.


CXC3
GeneSeq
WO9727299
Chemokines are a family of
Chemokine activities can be
Immune disorders



Accession

related small, secreted proteins
determined using assays known in



W23345

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138; Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


CCR7
GeneSeq
U.S. Pat. No. 6153441
Chemokines are a family of
Chemokine activities can be
Soluble CCR7 polypeptides



Accession B50859

related small, secreted proteins
determined using assays known in
may be useful for inhibiting





involved in biological processes
the art: Methods in Molecularr
chemokine activities and viral





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathlogies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Nerve Growth
GeneSeq
EP414151
Nerve Growth Factor
Proliferation assay using NR6R-
Neurological disorders, cancer


Factor-beta
Accession R11474


3T3 cells (Rizzino 1988 Cancer






Res. 48: 4266)


Nerve Growth
GeneSeq
EP859056
Nerve Growth Factor
Proliferation assay using NR6R-
Neurological disorders, cancer


Factor-beta2
Accession


3T3 cells (Rizzino 1988 Cancer



W69725


Res. 48: 4266)


Neurotrophin-3
GeneSeq
WO9821234
Neurotrophins regulate neuronal
Trk tyrosine kinase activation assays
Neurological disorders, cancer



Accession

cell survival and synaptic
known in the art can be used to



W48889

plasticity.
assay for neurotrophin activity, for






example, Proc Natl Acad Sci USA






2001 Mar 13; 98(6): 3555-3560.


Neurotrophin-4
GeneSeq
WO9325684
Neurotrophins regulate neuronal
Trk tyrosine kinase activation assays
Neurological disorders, cancer



Accession R47100

cell survival and synaptic
known in the art can be used to





plasticity.
assay for neurotrophin activity, for






example, Proc Natl Acad Sci USA






2001 Mar 13; 98(6): 3555-3560.


Neurotrophin-
GeneSeq
WO9325684
Neurotrophins regulate neuronal
Trk tyrosine kinase activation assays
Neurological disorders, cancer


4a
Accession R47101

cell survival and synaptic
known in the art can be used to





plasticity.
assay for neurotrophin activity, for






example, Proc Natl Acad Sci USA






2001 Mar 13; 98(6): 3555-3560.






13; 98(6): 3555-3560


Neurotrophin-
GeneSeq
WO9325684
Neurotrophins regulate neuronal
Trk tyrosine kinase activation assays
Neurological disorders, cancer


4b
Accession R47102

cell survival and synaptic
known in the art can be used to





plasticity.
assay for neurotrophin activity, for





tyrosine kinases.
example, Proc Natl Acad Sci USA






2001 Mar 13; 98(6): 3555-3560.


Neurotrophin-
GeneSeq
WO9325684
Neurotrophins regulate neuronal
Trk tyrosine kinase activation assays
Neurological disorders, cancer


4c
Accession R47103

cell survival and synaptic
known in the art can be used to





plasticity.
assay for neurotrophin activity, for






example, Proc Natl Acad Sci USA






2001 Mar 13; 98(6): 3555-3560.


Neurotrophin-
GeneSeq
WO9325684
Neurotrophins regulate neuronal
Trk tyrosine kinase activation assays
Neurological disorders, cancer


4d
Accession R47104

cell survival and synaptic
known in the art can be used to





plasticity.
assay for neurotrophin activity, for






example, Proc Natl Acad Sci USA






2001 Mar 13; 98(6): 3555-3560.


Platelet-Derived
GeneSeq
U.S. Pat. No. 5219739
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


Growth Factor
Accession R38918

Factor
using assays known in the art, such
proliferation of cells, such as


A chain



as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Hematopoietic and immune






example.
disorders. Antagonists may be







useful as anti-angiogenic







agents, and may be applicable







for cancer


Platelet-Derived
GeneSeq
U.S. Pat. No. 5219739
Vascular Endothelial Growth
VEGF activity can be determined
Promotion of growth and


Growth Factor
Accession R38919

Factor
using assays known in the art, such
proliferation of cells, such as


B chain



as those disclosed in International
vascular endothelial cells.






Publication No. WO0045835, for
Hematopoietic and immune






example.
disorders. Antagonists may be







useful as anti-angiogenic







agents, and may be applicable







for cancer


Stromal Derived
GeneSeq
WO9948528
Stromal Growth Factor
Proliferation assay using NR6R-
Hematopoietic, immune


Factor-1 alpha
Accession


3T3 cells (Rizzino 1988 Cancer
disorders, cancer



Y39995


Res. 48: 4266)


Stromal Derived
GeneSeq
CA2117953
Stromal Growth Factor
Proliferation assay using NR6R-
Hematopoietic, immune


Factor-1 beta
Accession R75420


3T3 cells (Rizzino 1988 Cancer
disorders, cancer






Res. 48: 4266)


Tarc
GeneSeq
WO9711969
Chemotactic for T lymphocytes.
Chemotactic leukocyte migration
Antiinflammatory, immune



Accession

May play a role in T-cell
assays are known in the art, for
disorders, cancer



W14917

development. Thought to bind
example: J. Immunol. Methods 33,





CCR8 and CCR4
((1980))


Prolactin
GeneSeq
WO9521625
Prolactin is involved in immune
Immune cell proliferation and
Reproductive system



Accession R78691

cell proliferation and apoptosis.
suppression of apoptosis by
disorders, cancer.






prolactin can be assayed by methods






well-known in the art, for example,






Buckley, A R and Buckley D J, Ann






N Y Acad Sci 2000; 917: 522-33,






and within.


Prolactin2
GeneSeq
U.S. Pat. No. 5955346
Prolactin is involved in immune
Immune cell proliferation and
Reproductive system



Accession

cell proliferation and apoptosis.
suppression of apoptosis by
disorders, cancer.



Y31764


prolactin can be assayed by methods






well-known in the art, for example,






Buckley, A R and Buckley D J, Ann






N Y Acad Sci 2000; 917: 522-33,






and within.


Follicle
GeneSeq
EP974359
FSH stimulates secretion of
FSH activities can be determined
Reproductive system


stimulating
Accession

interleukin-1 by cells isolated
using assays known in the art: J
disorders, cancer


hormone Alpha
Y54160

from women in the follicular
Gend Specif Med 1999 Nov-


subunit


phase
Dec; 2(6): 30-4; Mol Cell






Endocrinol. 1997 Nov






15; 134(2): 109-18.


Follicle
GeneSeq
EP974359
FSH stimulates secretion of
FSH activities can be determined
Reproductive system


stimulating
Accession

interleukin-1 by cells isolated
using assays known in the art: J
disorders, cancer


hormone Beta
Y54161

from women in the follicular
Gend Specif Med. 1999 Nov-


subunit


phase
Dec; 2(6): 30-4; Mol Cell






Endocrinol. 1997 Nov






15; 134(2): 109-18.


Substance P
GeneSeq
WO0054053
Substance P is associated with
Immunoregulation and bone marrow
diabetes mellitus,


(tachykinin)
Accession B23027

immunoregulation.
cell proliferation by substance P can
hypertension, cancer






be assayed by methods well-known






in the art, for example, Lai et al.






Proc Natl Acad Sci USA 2001 Mar






27; 98(7): 3970-5; Jallat-Daloz et al.






Allergy Asthma Proc 2001 Jan-Feb;






22(1): 17-23; Kahler et al. Exp Lung






Res 2001 Jan-Feb; 27(1): 25-46; and






Adamus MA and Dabrowski ZJ. J






Cell Biochem 2001; 81(3)499-506.


Oxytocin
GeneSeq
WO0053755
Oxytocin is involved in the
Oxytocin and prostaglandin E(2)
inflammatory disorders,


(Neurophysin I)
Accessions

induction of prostaglandin (E2)
release and Oxytocin (Ca2+)
immunologic disorders, cancer



B24085 and

release, as well as an increased
increase can be assayed by methods



B24086

amount of calcium release by
well-known in the art, for example,





smooth muscle cells.
Pavan et al., AM J Obset Gynecol






2000 Jul; 183(1): 76-82 and Holda et






al., Cell Calcium 1996 Jul; 20(1): 43-






51.


Vasopressin
GeneSeq
WO0053755
Vasopressin is believed to have a
Vasopressin activity can be
inflammatory disorders,


(Neurophysin
Accessions

direct antidiuretic action on the
determined using assays known in
immunologic disorders, cancer


II)
B24085 and

kidney, and it is thought to cause
the art, for example, Endocr Regul



B24086

vasoconstriction of the
1996 Mar; 30(1): 13-17.





peripheral vessels.


IL-1
GeneSeq
EP165654
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession P60326

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-1 mature
GeneSeq
EP456332
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R14855

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-1 beta
GeneSeq
WO9922763
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



Y08322

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-3 variants
GeneSeq
WO8806161
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



P80382, P80383,

synthesized by lymphocytes,
the art: Matthews et al., in



P80384, and

monocytes, and macrophages.
Lymphokines and Interferons: A



P80381

Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-4
GeneSeq
WO8702990
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession P70615

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &





cells, B cells, eosinophils, and
Mostowski (1990) J Immunol





lymphocytes), chemotaxis of
Methods 132, 287-295.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-4 muteins
GeneSeq
WO9747744
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W52151,

synthesized by lymphocytes,
the art: Matthews et al., in



W52152,

monocytes, and macrophages.
Lymphokines and Interferons: A



W52153,

Known functions include
Practical Approach, Clemens et al.,



W52154,

stimulating proliferation of
eds, IRL Press, Washington, D.C.



W52155,

immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &



W52156,

cells, B cells, eosinophils, and
Mostowski (1990) J Immunol



W52157,

lymphocytes), chemotaxis of
Methods 132, 287-295.



W52158,

neutrophils and T lymphocytes,



W52159,

and/or inhibition of interferons.



W52160,



W52161,



W52162,



W52163,



W52164, and



W52165


IL-1 alpha
GeneSeq
EP324447
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession P90108

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-3 variants
GeneSeq
WO9307171
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



R38561, R38562,

synthesized by lymphocytes,
the art: Matthews et al., in



R38563, R38564,

monocytes, and macrophages.
Lymphokines and Interferons: A



R38565, R38566,

Known functions include
Practical Approach, Clemens et al.,



R38567, R38568,

stimulating proliferation of
eds, IRL Press, Washington, D.C.



R38569, R38570,

immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et



R38571, and

cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-



R38572

lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-6
GeneSeq
WO9402512
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



R45717 and

synthesized by lymphocytes,
the art: Matthews et al., in



R45718

monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Aarden et al





cells, B cells, eosinophils, and
(1987) Eur. J. Immunol 17, 1411-





lymphocytes), chemotaxis of
16.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-13
GeneSeq
WO9404680
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R48624

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Boutelier et





cells, B cells, eosinophils, and
al (1995) J. Immunol. Methods





lymphocytes), chemotaxis of
181, 29.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-4 mutein
GeneSeq
DE4137333
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R47182

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &





cells, B cells, eosinophils, and
Mostowski (1990) J Immunol





lymphocytes), chemotaxis of
Methods 132, 287-295.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-4 mutein
GeneSeq
DE4137333
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Y124X
Accession R47183

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &





cells, B cells, eosinophils, and
Mostowski (1990) J Immunol





lymphocytes), chemotaxis of
Methods 132, 287-295.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-4 mutein
GeneSeq
DE4137333
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Y124G
Accession R47184

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &





cells, B cells, eosinophils, and
Mostowski (1990) J Immunol





lymphocytes), chemotaxis of
Methods 132, 287-295.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9317698
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin-10
Accession R41664

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


(precursor)


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Thompson-





cells, B cells, eosinophils, and
Snipes et al (1991) J. Exp. Med.





lymphocytes), chemotaxis of
173, 507-510.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9318783-A
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin 10
Accession R42642

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Thompson-





cells, B cells, eosinophils, and
Snipes et al (1991) J. Exp. Med.





lymphocytes), chemotaxis of
173, 507-510.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP569042
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-1
Accession R42447

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


beta precursor


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Interleukin-
GeneSeq
EP578278
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


1alpha
Accession R45364

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


MIRAP
GeneSeq
WO9117184
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R14400

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
JP04063595
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-3
Accession R22813

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
JP04063595
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-3
Accession R22814

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


variant


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-1i fragments
GeneSeq
EP541920
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



R35484 and

synthesized by lymphocytes,
the art: Matthews et al., in



R35485

monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-1 inhibitor
GeneSeq
EP541920
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


(IL-1i)
Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



R35486 and

synthesized by lymphocytes,
the art: Matthews et al., in



R35484

monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


ICE 22 kD
GeneSeq
EP533350
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


subunit
Accession R33780

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


ICE 20 kD
GeneSeq
EP533350
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


subunit
Accession R33781

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


ICE 10 kD
GeneSeq
EP533350
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


subunit
Accession R33782

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9317698
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin-10
Accession R41664

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


(precursor)


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Thompson-





cells, B cells, eosinophils, and
Snipes et al (1991) J. Exp. Med.





lymphocytes), chemotaxis of
173, 507-510.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9318783
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin 10
Accession R42642

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Thompson-





cells, B cells, eosinophils, and
Snipes et al (1991) J. Exp. Med.





lymphocytes), chemotaxis of
173, 507-510.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP569042-
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-1
Accession R42447

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


beta precursor


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9403492
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-6
Accession R49041

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Aarden et al





cells, B cells, eosinophils, and
(1987) Eur. J. Immunol 17, 1411-





lymphocytes), chemotaxis of
16.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Mutant
GeneSeq
WO9411402-
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin 6
Accession R54990

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


S176R


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Aarden et al





cells, B cells, eosinophils, and
(1987) Eur. J. Immunol 17, 1411-





lymphocytes), chemotaxis of
16.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Interleukin 6
GeneSeq
JP06145063
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R55256

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Aarden et al





cells, B cells, eosinophils, and
(1987) Eur. J. Immunol 17, 1411-





lymphocytes), chemotaxis of
16.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Interleukin 8
GeneSeq
JP06100595
Interleukins are a group of
Interleukin activity can be
Soluble IL-8 receptor


(IL-8) receptor
Accession R53932

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Holmes et al





cells, B cells, eosinophils, and
(1991) Science 253, 1278-80.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
U.S. Pat. No. 5328988
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-7
Accession R59919

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Park et al





cells, B cells, eosinophils, and
(1990) J. Exp. Med. 171, 1073-79.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-3 containing
GeneSeq
WO9521254
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


fusion protein
Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



R79342 and

synthesized by lymphocytes,
the art: Matthews et al., in



R79344

monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-3 mutant
GeneSeq
WO9521254
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


proteins
Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



R79254, R79255,

synthesized by lymphocytes,
the art: Matthews et al., in



R79256, R79257,

monocytes, and macrophages.
Lymphokines and Interferons: A



R79258, R79259,

Known functions include
Practical Approach, Clemens et al.,



R79260, R79261,

stimulating proliferation of
eds, IRL Press, Washington, D.C.



R79262, R79263,

immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et



R79264, R79265,

cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-



R79266, R79267,

lymphocytes), chemotaxis of
334.



R79268, R79269,

neutrophils and T lymphocytes,



R79270, R79271,

and/or inhibition of interferons.



R79272, R79273,



R79274, R79275,



R79276, R79277,



R79278, R79279,



R79280, R79281,



R79282, R79283,



R79284, and



R79285


IL-15
GeneSeq
ZA9402636
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R66927

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Giri et al





cells, B cells, eosinophils, and
(1994) EMBO J. 13 2822-2830.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-12 p40
GeneSeq
AU9466072
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


subunit.
Accession R63018

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


AGF
GeneSeq
WO9429344
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R64240

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9519786
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-12
Accession R79187

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


40 kD subunit


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9530695
Interleukins are a group of
Interleukin activity can be
Soluble IL-15 receptor


interleukin-15
Accession R90843

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


receptor from


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin


clone P1


monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Giri et al





cells, B cells, eosinophils, and
(1994) EMBO J. 13 2822-2830.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9604306-
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-7
Accession R92796

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Park et al





cells, B cells, eosinophils, and
(1990) J. Exp. Med. 171, 1073-79.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


interleukin-9
GeneSeq
WO9604306
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R92797

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


interleukin-3
GeneSeq
WO9604306
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R92801

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9604306
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-5
Accession R92802

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140, 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Recombinant
GeneSeq
DE19617202
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-16
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W33373

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lim et al





cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 2566-70.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-16
GeneSeq
DE19617202
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


protein
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W33234

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lim et al





cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 2566-70.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Thr117 human
GeneSeq
WO9708321
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin 9
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W27521

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Met117 human
GeneSeq
WO9708321
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin 9
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W27522

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
U.S. Pat. No. 5817306
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


intracellular IL-
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


1 receptor
W80777

synthesized by lymphocytes,
the art: Matthews et al., in


antagonist.


monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP864585
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-18
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


protein (IL-18)
W77158

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Ushio et al





cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 4274-79.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP861663
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin 18
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W77077

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Ushio et al





cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 4274-79.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP861663
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin 18
Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


derivatives
W77083,

synthesized by lymphocytes,
the art: Matthews et al., in



W77084,

monocytes, and macrophages.
Lymphokines and Interferons: A



W77085,

Known functions include
Practical Approach, Clemens et al.,



W77086,

stimulating proliferation of
eds, IRL Press, Washington, D.C.



W77087,

immune cells (e.g., T helper
1987, pp. 221-225; and Ushio et al



W77088, and

cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 4274-79.



W77089

lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Interleukin-9
GeneSeq
WO9827997
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


(IL-9) mature
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


protein (Thr117
W68158

synthesized by lymphocytes,
the art: Matthews et al., in


version).


monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-9 mature
GeneSeq
WO9827997
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


protein variant
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


(Met117
W68157

synthesized by lymphocytes,
the art: Matthews et al., in


version).


monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-9
GeneSeq
WO9824904
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


receptor protein
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


variant #3.
W64058

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-9
GeneSeq
WO9824904
Interleukins are a group of
Interleukin activity can be
Soluble IL-9 receptor


receptor protein
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


variant fragment
W64060

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-9
GeneSeq
WO9824904
Interleukins are a group of
Interleukin activity can be
Soluble IL-9 receptor


receptor protein
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


variant
W64061

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin


fragment#3.


monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9817689
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin-12
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


p40 protein
W51311

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9817689
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


Interleukin-12
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


p35 protein
W51312

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human protein
GeneSeq
DE19649233-
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


with IL-16
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


activity
W63753

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lim et al





cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 2566-70.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human protein
GeneSeq
DE19649233-
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


with IL-16
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


activity
W59425

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lim et al





cells, B cells, eosinophils, and
(1996) J. Immunol. 156, 2566-70.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
U.S. Pat. No. 5747024
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-15
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W53878

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Giri et al





cells, B cells, eosinophils, and
(1994) EMBO J. 13 2822-2830.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human wild-
GeneSeq
WO9747744
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


type interleukin-
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


4 (hIL-4)
W52149

synthesized by lymphocytes,
the art: Matthews et al., in


protein


monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &





cells, B cells, eosinophils, and
Mostowski (1990) J Immunol





lymphocytes), chemotaxis of
Methods 132, 287-295.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


interleukin-4
GeneSeq
WO9747744
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


muteins
Accessions

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W52150,

synthesized by lymphocytes,
the art: Matthews et al., in



W52151,

monocytes, and macrophages.
Lymphokines and Interferons: A



W52153,

Known functions include
Practical Approach, Clemens et al.,



W52154,

stimulating proliferation of
eds, IRL Press, Washington, D.C.



W52155,

immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &



W52156,

cells, B cells, eosinophils, and
Mostowski (1990) J Immunol



W52157,

lymphocytes), chemotaxis of
Methods 132, 287-295.



W52158,

neutrophils and T lymphocytes,



W52159,

and/or inhibition of interferons.



W52160,



W52161,



W52162,



W52163,



W52164,



W52165,



W52166, and



W52167


Human
GeneSeq
WO9935268
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin 1
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


delta
Y28408

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9935268
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-1
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


receptor
Y24395

synthesized by lymphocytes,
the art: Matthews et al., in


antagonist beta


monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human EDIRF
GeneSeq
WO9932632
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


II protein
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


sequence
Y22199

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human EDIRF
GeneSeq
WO9932632
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


I protein
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


sequence
Y22197

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-
GeneSeq
WO9919480
Interleukins are a group of
Interleukin activity can be
Soluble IL-IRD10 receptor


IRD10 protein
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


sequence
Y14131

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-
GeneSeq
WO9919480
Interleukins are a group of
Interleukin activity can be
Soluble IL-IRD9 receptor


IRD9
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



Y14122

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human DNAX
GeneSeq
WO9919491
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-40
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



Y09196

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


(DIL-40)
GeneSeq
WO9919491
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


alternative
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


sequence
Y09197

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-11
GeneSeq
WO9405318
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R50176

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lu et al





cells, B cells, eosinophils, and
(1994) J immunol. Methods 173, 19.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP566410
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


adipogenesis
Accession R43260

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


inhibitory factor


synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-11
GeneSeq
JP08127539
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W02202

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lu et al





cells, B cells, eosinophils, and
(1994) J immunol. Methods 173, 19.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-14
GeneSeq
WO9416074
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R55800

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Ambrus et al





cells, B cells, eosinophils, and
(1993) PNAS 90, 63330-34.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-17 receptor
GeneSeq
U.S. Pat. No. 6072033
Interleukins are a group of
Interleukin activity can be
Soluble IL-17 receptor



Accession B03807

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yao et al





cells, B cells, eosinophils, and
(1995) J. Immunol. 155, 5483-86.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-17
GeneSeq
WO9518826
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R76573

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yao et al





cells, B cells, eosinophils, and
(1995) J. Immunol. 155, 5483-86.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


CTLA-8
GeneSeq
WO9704097
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W13651

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-19
GeneSeq
WO9808870
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



W37935

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Gallagher et





cells, B cells, eosinophils, and
al (2000) Genes Immun. 1, 442-50.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-21 (TIF)
GeneSeq
WO0024758
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer



Y92879

synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Parrish-





cells, B cells, eosinophils, and
Novak et al (2000) Nature 408, 57-





lymphocytes), chemotaxis of
63.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-8 receptor
GeneSeq
WO9306229
Interleukins are a group of
Interleukin activity can be
Soluble IL-8 receptor



Accession R33420

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Holmes et al





cells, B cells, eosinophils, and
(1991) Science 253, 1278-80.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human type II
GeneSeq
U.S. Pat. No. 5464937
Interleukins are a group of
Interleukin activity can be
Soluble type II interleukin-1


interleukin-1
Accession R85480

multifunctional cytokines
determined using assays known in
receptor polypeptides may be


receptor


synthesized by lymphocytes,
the art: Matthews et al., in
useful for inhibiting





monocytes, and macrophages.
Lymphokines and Interferons: A
interleukin activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP638644
Interleukins are a group of
Interleukin activity can be
Soluble IL-12 receptor


interleukin-12
Accession R69632

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


receptor


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Interleukin 8
GeneSeq
U.S. Pat. No. 5440021
Interleukins are a group of
Interleukin activity can be
Soluble IL-8 receptor B


receptor B
Accession R80758

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Holmes et al





cells, B cells, eosinophils, and
(1991) Science 253, 1278-80.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-8
GeneSeq
JP08103276
Interleukins are a group of
Interleukin activity can be
Soluble IL-8 receptor A


receptor protein
Accession B09989

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


hIL8RA


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Holmes et al





cells, B cells, eosinophils, and
(1991) Science 253, 1278-80.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-8
GeneSeq
JP08103276
Interleukins are a group of
Interleukin activity can be
Soluble IL-8 receptor


receptor protein
Accession B09990

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


hIL8RB


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Holmes et al





cells, B cells, eosinophils, and
(1991) Science 253, 1278-80.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Interleukin-2
GeneSeq
WO9621732-
Interleukins are a group of
Interleukin activity can be
Soluble IL-2 receptor


receptor
Accession R97569

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


associated


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin


protein p43


monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Gillis et al





cells, B cells, eosinophils, and
(1978) J. Immunol. 120, 2027.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9629408
Interleukins are a group of
Interleukin activity can be
Soluble IL-17 receptor


interleukin-17
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


receptor
W04185

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yao et al





cells, B cells, eosinophils, and
(1995) J. Immunol. 155, 5483-86.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9619574
Interleukins are a group of
Interleukin activity can be
Soluble IL-11 receptor


interleukin-11
Accession R99090

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


receptor


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Lu et al





cells, B cells, eosinophils, and
(1994) J immunol. Methods 173, 19.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9623067
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,


interleukin-1
Accession

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer


receptor
W01911

synthesized by lymphocytes,
the art: Matthews et al., in


accessory


monocytes, and macrophages.
Lymphokines and Interferons: A


protein


Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


AGF Protein
GeneSeq
U.S. Pat. No. 5488032
Interleukins are a group of
Interleukin activity can be
inflammatory disorders,



Accession R92749

multifunctional cytokines
determined using assays known in
immunologic disorders, cancer





synthesized by lymphocytes,
the art: Matthews et al., in





monocytes, and macrophages.
Lymphokines and Interferons: A





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9607739
Interleukins are a group of
Interleukin activity can be
Soluble IL- type-3 receptor


interleukin-1
Accession R91064

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


type-3 receptor


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Orencole &





cells, B cells, eosinophils, and
Dinarello (1989) Cytokine 1, 14-20.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9720926
Interleukins are a group of
Interleukin activity can be
Soluble IL-13 beta receptor


interleukin-13
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


beta receptor
W24972

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Boutelier et





cells, B cells, eosinophils, and
al (1995) J. Immunol. Methods





lymphocytes), chemotaxis of
181, 29.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
WO9720926
Interleukins are a group of
Interleukin activity can be
Soluble IL-13 alpha receptor


interleukin-13
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


alpha receptor
W24973

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Boutelier et





cells, B cells, eosinophils, and
al (1995) J. Immunol. Methods





lymphocytes), chemotaxis of
181, 29.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
U.S. Pat. No. 5599905
Interleukins are a group of
Interleukin activity can be
Soluble IL-4 receptor


interleukin-4
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


receptor
W13499

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Siegel &





cells, B cells, eosinophils, and
Mostowski (1990) J. Immunol





lymphocytes), chemotaxis of
Methods 132, 287-295.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP759466
Interleukins are a group of
Interleukin activity can be
Soluble IL-12 beta-2 receptor


interleukin-12
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


beta-2 receptor
W12771

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human
GeneSeq
EP759466
Interleukins are a group of
Interleukin activity can be
Soluble IL-12 beta-1 receptor


interleukin-12
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


beta-1 receptor.
W12772

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Hori et al





cells, B cells, eosinophils, and
(1987), Blood 70, 1069-1078.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-9
GeneSeq
WO9824904
Interleukins are a group of
Interleukin activity can be
Soluble IL-9 receptor


receptor protein
Accessions

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



W64055,

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin



W64056, and

monocytes, and macrophages.
Lymphokines and Interferons: A
activities.



W64057

Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yang et al





cells, B cells, eosinophils, and
(1989) Blood 74, 1880-84.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-10 receptor
GeneSeq
U.S. Pat. No. 5716804
Interleukins are a group of
Interleukin activity can be
Soluble IL-10 receptor



Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



W41804

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Thompson-





cells, B cells, eosinophils, and
Snipes et al (1991) J. Exp. Med.





lymphocytes), chemotaxis of
173, 507-510.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-6
GeneSeq
JP11196867
Interleukins are a group of
Interleukin activity can be
Soluble IL-6 receptor


receptor
Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



Y30938

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Aarden et al





cells, B cells, eosinophils, and
(1987) Eur. J. Immunol 17, 1411-





lymphocytes), chemotaxis of
16.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Il-17 receptor
GeneSeq
U.S. Pat. No. 6096305
Interleukins are a group of
Interleukin activity can be
Soluble IL-17 receptor



Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



Y97181

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yao et al





cells, B cells, eosinophils, and
(1995) J. Immunol. 155, 5483-86.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Il-17 receptor
GeneSeq
U.S. Pat. No. 6100235
Interleukins are a group of
Interleukin activity can be
Soluble IL-17 receptor



Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



Y97131

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Yao et al





cells, B cells, eosinophils, and
(1995) J. Immunol. 155, 5483-86.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


human
GeneSeq
EP509826
Interleukins are a group of
Interleukin activity can be
Soluble IL-3 receptor


interleukin-3
Accession R25300

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


receptor


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human GM-
GeneSeq
WO9102063
Interleukins are a group of
Interleukin activity can be
Soluble GM-CSF receptor


CSF receptor
Accession R10919

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Human IL-5
GeneSeq
EP492214
Interleukins are a group of
Interleukin activity can be
Soluble IL-5 receptor alpha


receptor alpha
Accession R25064

multifunctional cytokines
determined using assays known in
polypeptides may be useful for


chain


synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140, 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Il-5 receptor
GeneSeq
WO9847923
Interleukins are a group of
Interleukin activity can be
Soluble IL-5 receptor



Accession

multifunctional cytokines
determined using assays known in
polypeptides may be useful for



W82842

synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Kitamura et





cells, B cells, eosinophils, and
al (1989) J Cell Physiol. 140, 323-





lymphocytes), chemotaxis of
334.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


Il-6 receptor
GeneSeq
JP05091892
Interleukins are a group of
Interleukin activity can be
Soluble IL-6 receptor



Accession R37215

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Aarden et al





cells, B cells, eosinophils, and
(1987) Eur. J. Immunol 17, 1411-





lymphocytes), chemotaxis of
16.





neutrophils and T lymphocytes,





and/or inhibition of interferons.


human B cell
GeneSeq
AU8928720
Interleukins are a group of
Interleukin activity can be
Soluble B cell stimulating


stimulating
Accession P90525

multifunctional cytokines
determined using assays known in
factor-2 receptor polypeptides


factor-2


synthesized by lymphocytes,
the art: Matthews et al., in
may be useful for inhibiting


receptor


monocytes, and macrophages.
Lymphokines and Interferons: A
interleukin activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225.





cells, B cells, eosinophils, and





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


IL-7 receptor
GeneSeq
EP403114
Interleukins are a group of
Interleukin activity can be
Soluble IL-7 receptor


clone
Accession R08330

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Park et al





cells, B cells, eosinophils, and
(1990) J. Exp. Med. 171, 1073-79.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


EPO receptor;
GeneSeq
WO9008822
EPO Receptor is involved in the
EPO Receptor activity can be
inflammatory disorders,


EPOR
Accession R06512

proliferation and differentiation
determined using assays known in
immunologic disorders,





of erythroblasts.
the art, such as, J Biol Chem 2001
cancer, erythroblast






Mar 23; 276(12): 8995-9002; JAK2
proliferation and






protein tyrosine kinase activity:
differentiation






Blood 1994 Sep 1; 84(5): 1501-7 and






Mol Cell Biol. 1994






Oct; 14(10): 6506-14.


IL-15 receptor
GeneSeq
WO9530695
Interleukins are a group of
Interleukin activity can be
Soluble IL-15 receptor



Accession R90843

multifunctional cytokines
determined using assays known in
polypeptides may be useful for





synthesized by lymphocytes,
the art: Matthews et al., in
inhibiting interleukin





monocytes, and macrophages.
Lymphokines and Interferons: A
activities.





Known functions include
Practical Approach, Clemens et al.,





stimulating proliferation of
eds, IRL Press, Washington, D.C.





immune cells (e.g., T helper
1987, pp. 221-225; and Giri et al





cells, B cells, eosinophils, and
(1994) EMBO J. 13 2822-2830.





lymphocytes), chemotaxis of





neutrophils and T lymphocytes,





and/or inhibition of interferons.


CD137; 4-IBB
GeneSeq
WO9507984
Activities associated with
Apoptosis activity, NF-kB
Soluble 4-IBB receptor


Receptor
Accession R70977

apoptosis, NF-kB activation,
activation, and B and T cell co-
polypeptides may be useful for


Protein


and co-stimulation of immune
stimulation can be determined using
inhibiting apoptosis, NF-kB





cells such as T and B cells.
assays known in the art: Moore et
activation, and/or co-






al., 1999, Science, 285(5425): 260-3;
stimulation of immune cells






Song HY etal., 1997 Proc Natl Acad
such as B and T cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


BCMA
GeneSeq
WO0068378
Activities associated with
Apoptosis activity, NF-kB
Soluble BCMA receptor



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
polypeptides may be useful for



Y71979

and co-stimulation of immune
stimulation can be determined using
inhibiting apoptosis, NF-kB





cells such as T and B cells.
assays known in the art: Moore et
activation, and/or co-






al., 1999, Science, 285(5425): 260-3;
stimulation of immune cells






Song HY etal., 1997 Proc Natl Acad
such as B and T cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


CD27
GeneSeq
WO9201049
Activities associated with
Apoptosis activity, NF-kB
Soluble CD27 polypeptides



Accession R20814

apoptosis, NF-kB activation,
activation, and B and T cell co-
may be useful for inhibiting





and co-stimulation of immune
stimulation can be determined using
apoptosis, NF-kB activation,





cells such as T and B cells.
assays known in the art: Moore et
and/or co-stimulation of






al., 1999, Science, 285(5425): 260-3;
immune cells such as B and T






Song HY etal., 1997 Proc Natl Acad
cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


CD30
GeneSeq
DE4200043
Activities associated with
Apoptosis activity, NF-kB
Soluble CD30 polypeptides



Accession R35478

apoptosis, NF-kB activation,
activation, and B and T cell co-
may be useful for inhibiting





and co-stimulation of immune
stimulation can be determined using
apoptosis, NF-kB activation,





cells such as T and B cells.
assays known in the art: Moore et
and/or co-stimulation of






al., 1999, Science, 285(5425): 260-3;
immune cells such as B and T






Song HY etal., 1997 Proc Natl Acad
cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


CD40
GeneSeq
WO9945944
Activities associated with
Apoptosis activity, NF-kB
Soluble CD40 polypeptides



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
may be useful for inhibiting



Y33499

and co-stimulation of immune
stimulation can be determined using
apoptosis, NF-kB activation,





cells such as T and B cells.
assays known in the art: Moore et
and/or co-stimulation of






al., 1999, Science, 285(5425): 260-3;
immune cells such as B and T






Song HY etal., 1997 Proc Natl Acad
cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


EDAR
Genbank

Activities associated with
Apoptosis activity, NF-kB
Immune Disorders,



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
Lymphomas, X-linked



AAD50077

and co-stimulation of immune
stimulation can be determined using
hypohidrotic ectodermal





cells such as T and B cells.
assays known in the art: Moore et
dysplasia






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


OX40; ACT-4
GeneSeq
WO9512673
Activities associated with
Apoptosis activity, NF-kB
Immune Disorders,



Accession R74737

apoptosis, NF-kB activation,
activation, and B and T cell co-
Lymphomas, T cell disorders





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


TACI
GeneSeq
WO9839361
Activities associated with
Apoptosis activity, NF-kB
Soluble TACI receptor



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
polypeptides may be useful for



W75783

and co-stimulation of immune
stimulation can be determined using
inhibiting apoptosis, NF-kB





cells such as T and B cells.
assays known in the art: Moore et
activation, and/or co-






al., 1999, Science, 285(5425): 260-3;
stimulation of immune cells






Song HY etal., 1997 Proc Natl Acad
such as B and T cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


TNF-R
GeneSeq
AU9058976
Activities associated with
Apoptosis activity, NF-kB
Soluble TNF-R receptor



Accession R10986

apoptosis, NF-kB activation,
activation, and B and T cell co-
polypeptides may be useful for





and co-stimulation of immune
stimulation can be determined using
inhibiting apoptosis, NF-kB





cells such as T and B cells.
assays known in the art: Moore et
activation, and/or co-






al., 1999, Science, 285(5425): 260-3;
stimulation of immune cells






Song HY etal., 1997 Proc Natl Acad
such as B and T cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


TNF-RII; TNF
GeneSeq
EP418014
Activities associated with
Apoptosis activity, NF-kB
Soluble TNFR-II receptor


p75 Receptor;
Accession R11141

apoptosis, NF-kB activation,
activation, and B and T cell co-
polypeptides may be useful for


Death Receptor


and co-stimulation of immune
stimulation can be determined using
inhibiting apoptosis, NF-kB





cells such as T and B cells.
assays known in the art: Moore et
activation, and/or co-






al., 1999, Science, 285(5425): 260-3;
stimulation of immune cells






Song HY etal., 1997 Proc Natl Acad
such as B and T cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


hAPO-4; TROY
GeneSeq
WO9911791
Activities associated with
Apoptosis activity, NF-kB
Immune Disorders, Cancers



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-



W93581

and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


TNF-alpha
GeneSeq
EP205038
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,


precursor
Accession P60074

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


Human TNF-
GeneSeq
EP619372
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,


alpha
Accession R62463

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


Human TNF-
GeneSeq
EP563714
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,


alpha
Accession R42679

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


Human TNF-
GeneSeq
WO0064479
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,


beta (LT-alpha)
Accession B37799

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


LT-alpha
GeneSeq
EP250000
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession P70107

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


LT-beta
GeneSeq
WO9413808
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession R56869

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


OPGL
GeneSeq
WO9846751
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders,



W83195

and co-stimulation of immune
stimulation can be determined using
cancer, loss of bone mass.





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


FasL
GeneSeq
WO9903999
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer



W98071

and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


FasL
GeneSeq
WO9903998
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer



W95041

and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


CD27L
GeneSeq
WO9405691
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession R50121

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


CD30 ligand
GeneSeq
WO9324135
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession R45007

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


CD40L
GeneSeq
WO9529935
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession R85486

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


4-IBB ligand
GeneSeq
U.S. Pat. No. 5674704
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer



W26657

and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


FAS Ligand
GeneSeq
WO0058465
Activities associated with
Apoptosis activity, NF-kB
Soluble DcR3 polypeptides


Inhibitory
Accession B19335

apoptosis, NF-kB activation,
activation, and B and T cell co-
may be useful for inhibiting


Protein (DcR3)


and co-stimulation of immune
stimulation can be determined using
apoptosis, NF-kB activation,





cells such as T and B cells.
assays known in the art: Moore et
and/or co-stimulation of






al., 1999, Science, 285(5425): 260-3;
immune cells such as B and T






Song HY etal., 1997 Proc Natl Acad
cells.






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


OX40L
GeneSeq
WO9521915
Activities associated with
Apoptosis activity, NF-kB
inflammatory disorders,



Accession R79903

apoptosis, NF-kB activation,
activation, and B and T cell co-
immunologic disorders, cancer





and co-stimulation of immune
stimulation can be determined using





cells such as T and B cells.
assays known in the art: Moore et






al., 1999, Science, 285(5425): 260-3;






Song HY etal., 1997 Proc Natl Acad






Sci USA 94(18): 9792-6; Epsevik






and Nissen-Meyer, 1986, J.






Immunol. Methods.


Protease
GeneSeq
WO9106561
Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


inhibitor
Accessions

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,


peptides
R12435, R12436,


EP0387231. One can modify the
cancer, viral infections



R12437, R12438,


assay to look for inhibition using



R12439, R12440,


any of the disclosed protease



and R1244


inhibitor polypeptides.


Retroviral
GeneSeq
EP387231
Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


protease
Accessions

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,


inhibitors
R06660, R06661,


EP0387231. One can modify the
cancer, viral infections



R06662, R06663,


assay to look for inhibition using



R06664, R06665,


any of the disclosed protease



R06666, R06667,


inhibitor polypeptides.



R06668, R06669,



R06670, R06671,



R06672, R06673,



R06674, R06675,



and R06676


HIV protease
GeneSeq
WO9301828
Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


inhibiting
Accessions

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,


peptides
R59293, R59294,


EP0387231. One can modify the
cancer, viral infections



R59295, R59296,


assay to look for inhibition using



R59297,


any of the disclosed protease



R59298, R59299,


inhibitor polypeptides.



R59300, R59301,



R59302, R59301,



R59302, R59303,



R59304, R59305,



R59306, R59307,



R59308, R59309,



R59310, R59311,



R59312, R59313,



R59314, R59315,



R59316, R59317,



R59318, R59319,



R59320, R59321,



R59322, R59323,



R59324, R59325,



R59326, R59327,



R59328, R59329,



R59330, R59331,



R59332, R59333,



R59334, R59335,



R59336, R59337,



R59338, R59339,



R59340, R59341,



R59342, R59343,



R59344, R59345,



R59346, R59347,



R59348, R59349,



and R59350


HIV-1 protease
GeneSeq
DE4412174
Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


inhibitors
Accessions

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,



R86326, R86327,


EP0387231. One can modify the
cancer, viral infections



R86328, R86329,


assay to look for inhibition using



R86330, R86331,


any of the disclosed protease



R86332, R86333,


inhibitor polypeptides.



R86334, R86335,



R86336, R86337,



R86338, R86339,



R86340, R86341,



R86342, R86343,



R86344, R86345,



R86346, R86347,



R86348, R86349,



R86350, R86351,



R86352, R86353,



R86354, R86355,



R86356, R86357,



R86358, R86359,



R86360, R86361,



R86362, R86363,



R86364, R86365,



R86366, R86367,



R86368, R86369,



R86370, and



R86371


HIV Inhibitor
GeneSeq
WO9959615
Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


Peptide
Accession

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,



Y89687


EP0387231. One can modify the
cancer, viral infections






assay to look for inhibition using






any of the disclosed protease






inhibitor polypeptides.


HIV Inhibitor
GeneSeq
WO9948513
Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


Peptide
Accession

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,



Y31955


EP0387231. One can modify the
cancer, viral infections






assay to look for inhibition using






any of the disclosed protease






inhibitor polypeptides.


HIV Inhibitor
www.sciencex

Peptides that inhibit the
HIV protease activities are known in
HIV, inflammatory disorders,


Peptide
press.org;

function/binding of HIV
the art: HIV protease assays:
immunologic disorders,



Published


EP0387231. One can modify the
cancer, viral infections



Online 12


assay to look for inhibition using



January 2001;


any of the disclosed protease



10.1126/scienc


inhibitor polypeptides.



e.1057453


Human
GeneSeq
WO9509232
Chemokines are a family of
Chemokine activities can be
Immune disorders, particularly


monocyte
Accession R73915

related small, secreted proteins
determined using assays known in
useful for treating bacterial


chemoattractant


involved in biological processes
the art: Methods in Molecular
and/or viral menigitis


factor hMCP-3


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9509232
Chemokines are a family of
Chemokine activities can be
Immune disorders, particularly


monocyte
Accession R73914

related small, secreted proteins
determined using assays known in
useful for treating bacterial


chemoattractant


involved in biological processes
the art: Methods in Molecular
and/or viral menigitis


factor hMCP-1


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human gro-beta
GeneSeq
WO9429341
Chemokines are a family of
Chemokine activities can be
Immune disorders,


chemokine
Accessions

related small, secreted proteins
determined using assays known in
inflammatory disorders, blood-



R66699 and

involved in biological processes
the art: Methods in Molecular
related disorders, stem cell



W17671

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplantation, cancer





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human gro-
GeneSeq
WO9429341
Chemokines are a family of
Chemokine activities can be
Immune disorders,


gamma
Accessions

related small, secreted proteins
determined using assays known in
inflammatory disorders, blood-


chemokine
R66700 and

involved in biological processes
the art: Methods in Molecular
related disorders, stem cell



W17672

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplantation, cancer





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human gro-
GeneSeq
WO9429341
Chemokines are a family of
Chemokine activities can be
Immune disorders,


alpha
Accessions

related small, secreted proteins
determined using assays known in
inflammatory disorders, blood-


chemokine
R66698 and

involved in biological processes
the art: Methods in Molecular
related disorders, stem cell



W18024

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplantation, cancer





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9632481
Chemokines are a family of
Chemokine activities can be
Immune disorders, partcularly


eosinophil-
Accession

related small, secreted proteins
determined using assays known in
treatment of eosinophilia,


expressed
WO5186

involved in biological processes
the art: Methods in Molecular
inflammation, allergies,


chemokine


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
asthma, leukaemia and


(EEC)


angiogenesis, and leukocyte
Protocols: Edited by: A. E. I.
lymphoma





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein PF4-414
Accessions

related small, secreted proteins
determined using assays known in
disorders, particularly


Full-Length and
R92318 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Mature
R99809

ranging from hematopoiesis,
Biology, 2000 vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot. T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa; NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein IL-8M3
Accession R99812

related small, secreted proteins
determined using assays known in
disorders, particularly





involved in biological processes
the art: Methods in Molecular
myelosuppression





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


interleukin-8
Accession R99814

related small, secreted proteins
determined using assays known in
disorders, particularly


(IL-8)


involved in biological processes
the art: Methods in Molecular
myelosuppression





ranging from hematopoiesis,
Biology, 2000, vol. 138; Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein IL-8M1
Accessions

related small, secreted proteins
determined using assays known in
disorders, particularly


Full-Length and
R99815 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Mature
R99803

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Scicnce 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein IL-8M8
Accessions

related small, secreted proteins
determined using assays known in
disorders, particularly


Full-Length and
R99816 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Mature
R99805

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253. 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein IL-8M9
Accessions

related small, secreted proteins
determined using assays known in
disorders, particularly


Full-Length and
R99817 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Mature
R99806

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein IL-
Accessions

related small, secreted proteins
determined using assays known in
disorders. particularly


8M10 Full-
R99818 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Length and
R99804

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


Mature


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein PF4-M2
Accessions

related small, secreted proteins
determined using assays known in
disorders, particularly


Full-Length and
R99819 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Mature
R99807

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine-like
GeneSeq
WO9613587
Chemokines are a family of
Chemokine activities can be
Cancer and blood-related


protein PF4-426
Accessions

related small, secreted proteins
determined using assays known in
disorders, particularly


Full-Length and
R99822 and

involved in biological processes
the art: Methods in Molecular
myelosuppression


Mature
R99811

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human foetal
GeneSeq
WO9622374
Chemokines are a family of
Chemokine activities can be
Immune disorders


spleen
Accession R98499

related small, secreted proteins
determined using assays known in


expressed


involved in biological processes
the art: Methods in Molecular


chemokine,


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


FSEC


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Liver expressed
GeneSeq
WO9616979
Chemokines are a family of
Chemokine activities can be
Inflammation of the liver


chemokine-1
Accession R95689

related small, secreted proteins
determined using assays known in


(LVEC-1)


involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Liver expressed
GeneSeq
WO9616979
Chemokines are a family of
Chemokine activities can be
Inflammation of the liver


chemokine-2
Accession R95690

related small, secreted proteins
determined using assays known in


(LVEC-2)


involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138; Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Pituitary
GeneSeq
WO9616979
Chemokines are a family of
Chemokine activities can be
Inflammation, particularly of


expressed
Accession R95691

related small, scereted proteins
determined using assays known in
the liver


chemokine


involved in biological processes
the art: Methods in Molecular


(PGEC)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Adenoid-
GeneSeq
WO9617868
Chemokines are a family of
Chemokine activities can be
Inflammation, angiogenesis,


expressed
Accession R97664

related small, secreted proteins
determined using assays known in
tumorigenesis,


chemokine


involved in biological processes
the art: Methods in Molecular
musculoskeletal disorders


(ADEC)


ranging from hematopoiesis,
Biology, 2000, vol. 138. Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9741230
Chemokines are a family of
Chemokine activities can be
Immune disorders, cell


chemokine CC-
Accession

related small, secreted proteins
determined using assays known in
migration, proliferation, and


2
W38170

involved in biological processes
the art: Methods in Molecular
differentiation disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138; Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9741230
Chemokines are a family of
Chemokine activities can be
Immune disorders, cell


chemokine
Accession

related small, secreted proteins
determined using assays known in
migration, proliferation, and


HCC-1
W38171

involved in biological processes
the art: Methods in Molecular
differentiation disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9741230
Chemokines are a family of
Chemokine activities can be
Immune disorders, cell


chemokine CC-
Accession

related small, secreted proteins
determined using assays known in
migration, proliferation, and


3
W38172

involved in biological processes
the art: Methods in Molecular
differentiation disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Novel beta-
GeneSeq
WO9739126
Chemokines are a family of
Chemokine activities can be
Immune disorders, vascular


chemokine,
Accession

related small, secreted proteins
determined using assays known in
disorders, cancer


designated
W27271

involved in biological processes
the art: Methods in Molecular


PTEC


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, und leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human CX3C
GeneSeq
WO9727299
Chemokines are a family of
Chemokine activities can be
Immune disorders,


111 amino acid
Accession

related small, secreted proteins
determined using assays known in
inflammatory diseases,


chemokine
W23344

involved in biological processes
the art: Methods in Molecular
abnormal proliferation,





ranging front hematopoiesis,
Biology, 2000, vol. 138; Chemokine
regeneration, degeneration,





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and atrophy





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human CCF18
GeneSeq
WO9721812
Chemokines are a family of
Chemokine activities can be
Abnormal physiology and


chemokine
Accession

related small, secreted proteins
determined using assays known in
development disorders, can



W25942

involved in biological processes
the art: Methods in Molecular
also be used as an anti-viral





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
agent





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human beta-
GeneSeq
WO9725427
Chemokines are a family of
Chemokine activities can be
Chemotaxis, blood-related


chemokine
Accession

related small, secreted proteins
determined using assays known in
disorders, viral infection, HIV,


H1305 (MCP-2)
W26655

involved in biological processes
the art: Methods in Molecular
wound healing, cancer





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9712914
Chemokines are a family of
Chemokine activities can be
Inflammatory and immune


eosinocyte CC
Accession

related small, secreted proteins
determined using assays known in
disorders


type chemokine
W14990

involved in biological processes
the art: Methods in Molecular


eotaxin


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human thymus
GeneSeq
WO9711969
Chemokines are a family of
Chemokine activites can be
Inflammatory and immune


and activation
Accession

related small, secreted proteins
determined using assays known in
disorders


regulated
W14917

involved in biological processes
the art: Methods in Molecular


cytokine


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


(TARC)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9712041
Chemokines are a family of
Chemokine activities can be
Cancer, wound healing,


chemokine beta-
Accession

related small, secreted proteins
determined using assays known in
immune disorders


8 short forms
W16315

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Macrophage
GeneSeq
WO9640923
Chemokines are a family of
Chemokine activities can be
Inflammatory diseases, wound


derived
Accession

related small, secreted proteins
determined using assays known in
healing, angiogenesis


chemokine,
W20058

involved in biological processes
the art: Methods in Molecular


MDC


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9844117
Chemokines are a family of
Chemokine activities can be
Inflammatory and immune


chemokine
Accession

related small, secreted proteins
determined using assays known in
diseases


ZSIG-35
W30565

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Primate CC
GeneSeq
WO9832858
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


chemokine
Accession

related small, secreted proteins
determined using assays known in
disorders, abnormal


“ILINCK”
W69990

involved in biological processes
the art: Methods in Molecular
proliferation, regeneration,





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
generation and atrophy





angiogenesis, and leukocyte
Protocol. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Primate CXC
GeneSeq
WO9832858
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


chemokine
Accession

related small, secreted proteins
determined using assays known in
disorders, abnormal


“IBICK”
W69989

involved in biological processes
the art: Methods in Molecular
proliferation, regeneration,





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
generation and atrophy





angiogenesis, and leukocyte
Protocol. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human CC-type
GeneSeq
WO9831809
Chemokines are a family of
Chemokine activities can be
Immune, Inflammatory, and


chemokine
Accession

related small, secreted proteins
determined using assays known in
infectious disorders, cancer


protein
W69163

involved in biological processes
the art: Methods in Molecular


designated SLC


ranging from hematopoiesis,
Biology, 20O0, vol. 138. Chemokine


(secondary


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.


lymphoid


trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.


chemokine)


family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human CC
GeneSeq
WO9826071
Chemokines are a family of
Chemokine activities can be
Cancer and infectious diseases,


chemokine ELC
Accession

related small, secreted proteins
determined using assays known in
particularly herpes virus


protein
W62542

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human DVic-1
GeneSeq
WO9823750
Chemokines are a family of
Chemokine activities can be
Abnormal proliferation,


C-C chemokine
Accession

related small, secreted proteins
determined using assays known in
regeneration, degeneration,



W60649

involved in biological processes
the art: Methods in Molecular
and atrophy disorders,





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
including cancer





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines bave been





described, which bind to  ̃17





receptors thus far identified.


Human C-C
GeneSeq
WO9823750
Chemokines are a family of
Chemokine activities can be
Immune disorders, cell


chemokine
Accession

related small, secreted proteins
determined using assays known in
proliferation disorders, cancer


DGWCC
W60650

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human STCP-1
GeneSeq
WO9824907
Chemokines are a family of
Chemokine activities can be
Immune disorders, particularly



Accession

related small, secreted proteins
determined using assays known in
T cell related disorders, viral



W62783

involved in biological processes
the art: Methods in Molecular
infection, and inflammation,





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
especially joint





angiogenesis, and leukocyte
Protocols. Edited by; A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Exodus protein
GeneSeq
WO9821330
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory



Accession

related small, secreted proteins
determined using assays known in
disorders, angiogenesis,



W61279

involved in biological processes
the art: Methods in Molecular
cancer, and proliferation





ranging from hematopoiesis,
Biology, 2000. vol. 138: Chemokine
disorders, particularly





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
myeloproliferative diseases





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9814581
Chemokines are a family of
Chemokine activities can be
Cancer and degenerative


Chr19Kine
Accession

related small, secreted proteins
determined using assays known in
disorders


protein
W50887

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a slmilarly
Power.  ® Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human T cell
GeneSeq
U.S. Pat. No. 5780268
Chemokines are a family of
Chemokine activities can be
Immune, inflammatory, and


mixed
Accession

related small, secreted proteins
determined using assays known in
infectious disorders, cancer


lymphocyte
W58703

involved in biological processes
the art: Methods in Molecular


reaction


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


expressed


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.


chemokine


trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.


(TMEC)


family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human 6CKine
GeneSeq
WO9814581
Chemokines are a family of
Chemokine activities can be
Cancer and degenerative


protein
Accession

related small, secreted proteins
determined using assays known in
disorders



W50855

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


human liver and
GeneSeq
WO9817800
Chemokines are a family of
Chemokine activities can be
Immune, inflammatory, and


activation
Accession

related small, secreted proteins
determined using assays known in
infectious disorders, cancer


regulated
W57475

involved in biological processes
the art: Methods in Molecular


chemokine


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


(LARC)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


RANTES
GeneSeq
WO9744462
Chemokines are a family of
Chemokine activities can be
Infectious diseases,


peptide
Accession

related small, secreted proteins
determined using assays known in
particularly HIV



W29538

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138; Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot; T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


RANTES 8-68
GeneSeq
WO9744462
Chemokines are a family of
Chemokine activities can be
infectious diseases,



Accession

related small, secreted proteins
determined using assays known in
particularly HIV



W29529

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Prolocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identitied.


RANTES 9-68
GeneSeq
WO9744462
Chemokines are a family of
Chemokine activities can be
Infectious diseases,



Accession

related small, secreted proteins
determined using assays known in
particularly HIV



W29528

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9811226
Chemokines are a family of
Chemokine activities can be
Abnormal proliferation,


chemokine
Accession

related small, secreted proteins
determined using assays known in
regeneration, degeneration or


protein 331D5
W59433

involved in biological processes
the art: Methods in Molecular
atrophy, including cancer





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9811226
Chemokines are a family of
Chemokine activities can be
Abnormal proliferation,


chemokine
Accession

related small, secreted proteins
determined using assays known in
regeneration, degeneration or


protein 61164
W59430

involved in biological processes
the art: Methods in Molecular
atrophy, including cancer





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9809171
Chemokines are a family of
Chemokine activities can be
Immune, inflammatory, and


MCP-4
Accession

related small, secreted proteins
determined using assays known in
infectious diseases



W56690

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human stromal
GeneSeq
FR2751658
Chemokines are a family of
Chemokine activities can be
HIV infections


cell-derived
Accession

related small, secreted proteins
determined using assays known in


chemokine,
W50766

involved in biological processes
the art: Methods in Molecular


SDF-1


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Thymus
GeneSeq
WO9801557
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


expressed
Accession

related small, secreted proteins
determined using assays known in
disorders


chemokine
W44397

involved in biological processes
the art: Methods in Molecular


(TECK)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9801557
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


chemokine
Accession

related small, secreted proteins
determined using assays known in
disorders


MIP-3alpha
W44398

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9801557
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


chemokine
Accession

related small, secreted proteins
determined using assays known in
disorders


MIP-3beta
W44399

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9802459
Chemokines are a family of
Chemokine activities can be
Immune disorders, respiratory


monocyte
Accession

related small, secreted proteins
determined using assays known in
disorders, cancer


chemotactic
W42072

involved in biological processes
the art: Methods in Molecular


proprotein


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


(MCPP)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.


sequence


trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Macrophage-
GeneSeq
U.S. Pat. No. 5688927/
Chemokines are a family of
Chemokine activities can be
Immune, and inflammatory


derived
Accessions
U.S. Pat. No. 5932703
related small, secreted proteins
determined using assays known in
disorders, cancer


chemokine
W40811 and

involved in biological processes
the art: Methods in Molecular


(MDC)
Y24414

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Macrophage
GeneSeq
U.S. Pat. No. 5932703
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


derived
Accession

related small, secreted proteins
determined using assays known in
disorders


chemokine
Y24416

involved in biological processes
the art: Methods in Molecular


analogue MDC-


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


eyfy


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Macrophage
GeneSeq
U.S. Pat. No. 5932703
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


derived
Accession

related small, secreted proteins
determined using assays known in
disorders


chemokine
Y24413

involved in biological processes
the art: Methods in Molecular


analogue MDC


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


(n+1)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Macrophage
GeneSeq
U.S. Pat. No. 5932703
Chemokines are a family of
Chemokine activities can be
Immune and inflammatory


derived
Accession

related small, secreted proteins
determined using assays known in
disorders


chemokine
Y24415

involved in biological processes
the art: Methods in Molecular


analogue MDC-


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


yl


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human type CC
GeneSeq
JP11243960
Chemokines are a family of
Chemokine activities can be
Allergic diseases and HIV


chemokine
Accession

related small, secreted proteins
determined using assays known in
infection


eotaxin 3
Y43178

involved in biological processes
the art: Methods in Molecular


protein


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


sequence


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human MCP-3
GeneSeq
WO9946392
Chemokines are a family of
Chemokine activities can be
Cancer and immune disorders,


and human
Accession

related small, secreted proteins
determined using assays known in
particularly HIV infection


Muc-1 core
Y29893

involved in biological processes
the art: Methods in Molecular


epitope (VNT)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


fusion protein


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human IP-10
GeneSeq
WO9946392
Chemokines are a family of
Chemokine activities can be
Cancer and immune disorders,


and human
Accession

related small, secreted proteins
determined using assays known in
particularly HIV infection


Muc-1 core
Y29894

involved in biological processes
the art: Methods in Molecular


epitope (VNT)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


fusion protein


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human IP-10
GeneSeq
WO9946392
Chemokines are a family of
Chemokine activities can be
Cancer and immune disorders,


and HIV-1
Accession

related small, secreted proteins
determined using assays known in
particularly HIV infection


gp120
Y29897

involved in biological processes
the art: Methods in Molecular


hypervariable


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


region fusion


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.


protein


trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9936540
Chemokines are a family of
Chemokine activities can be
Breast disease, including


mammary
Accessions

related small, secreted proteins
determined using assays known in
cancer


associated
Y29092 and

involved in biological processes
the art: Methods in Molecular


chemokine
Y29093

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


(MACK)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.


protein Full-


trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.


Length and


family are involved in a similarly
Power.  © Humana Press Inc.,


Mature


diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Tim-1 protein
GeneSeq
WO9933990
Chemokines are a family of
Chemokine activities can be
Inflammation due to stimuli



Accession

related small, secreted proteins
determined using assays known in
such as heart attacks and



Y28290

involved in biological processes
the art: Methods in Molecular
stroke, infection, physical





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
trauma, UV or ionizing





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
radiation, burns, frostbite or





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
corrosive chemicals





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human Lkn-1
GeneSeq
WO9928473 and
Chemokines are a family of
Chemokine activities can be
HIV infection and cancer,


Full-Length and
Accessions
WO9928472
related small, secreted proteins
determined using assays known in
particularly leukemia


Mature protein
Y17280, Y17274,

involved in biological processes
the art: Methods in Molecular



Y17281, and

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine



Y17275

angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


N-terminal
GeneSeq
WO9920759
Chemokines are a family of
Chemokine activities can be
Inhibit or stimulate


modified
Accession

related small, secreted proteins
determined using assays known in
angiogenesis, inhibit the


chemokine met-
Y05818

involved in biological processes
the art: Methods in Molecular
binding of HIV


hSDF-1 alpha


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


N-terminal
GeneSeq
WO9920759
Chemokines are a family of
Chemokine activities can be
Inhibit or stimulate


modified
Accession

related small, secreted proteins
determined using assays known in
angiogenesis, inhibit the


chemokine met-
Y05819

involved in biological processes
the art: Methods in Molecular
binding of HIV,


hSDF-1 beta


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
antiinflammatory;





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
immunosuppressant





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


N-terminal
GeneSeq
WO9920759
Chemokines are a family of
Chemokine activities can be
Inhibit or stimulate


modified
Accession

related small, secreted proteins
determined using assays known in
angiogenesis, inhibit the


chemokine
Y05820

involved in biological processes
the art: Methods in Molecular
binding of HIV,


GroHEK/hSDF-


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
antiinflammatory;


1alpha


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
immunosuppressant





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


N-terminal
GeneSeq
WO9920759
Chemokines are a family of
Chemokine activities can be
Inhibit or stimulate


modified
Accession

related small, secreted proteins
determined using assays known in
angiogenesis, inhibit the


chemokine
Y05821

involved in biological processes
the art: Methods in Molecular
binding of HIV,


GroHEK/hSDF-


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
antiinflammatory;


1beta.


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
immunosuppressant





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Increase or enhance an


Eotaxin.
Accession

related small, secreted proteins
determined using assays known in
inflammatory response, an



Y14230

involved in biological processes
the art: Methods in Molecular
immune response or





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
haematopoietic cell-associated





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
activity; treat a vascular





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
indication; Cancer; enhance





family are involved in a similarly
Power.  © Humana Press Inc.,
wound healing, to prevent or





diverse range of pathologies
Totowa, NJ
treat asthma, organ transplant





including inflammation, allergy,

rejection, rheumatoid arthritis





tissue rejection, viral infection,

or allergy





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


hMCP1a
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14225

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response,





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


hMCP1b
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14226

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response,





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


hSDF1b
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14228

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine hIL-
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


8
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14229

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


hMCP1
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14222

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


hMCP2
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14223

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Chemokine
GeneSeq
WO9912968
Chemokines are a family of
Chemokine activities can be
Immune disorders, Vascular


hMCP3
Accession

related small, secreted proteins
determined using assays known in
disorders, Wound healing,



Y14224

involved in biological processes
the art: Methods in Molecular
cancer, prevent organ





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
transplant rejection, Increase





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
or enhance an inflammatory





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
response





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


C-C chemokine,
GeneSeq
EP905240
Chemokines are a family of
Chemokine activities can be
Inflammatory, immune and


MCP2
Accession

related small, secreted proteins
determined using assays known in
infectious diseases; pulmonary



Y05300

involved in biological processes
the art: Methods in Molecular
diseases and skin disorders;





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
tumours, and angiogenesis-





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and haematopoiesis-related





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Wild type
GeneSeq
EP906954
Chemokines are a family of
Chemokine activities can be
Inflammatory, immune and


monocyte
Accession

related small, secreted proteins
determined using assays known in
infectious diseases; pulmonary


chemotactic
Y07233

involved in biological processes
the art: Methods in Molecular
diseases and skin disorders;


protein 2


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
tumours, and angiogenesis-





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and haematopoiesis-related





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Truncated
GeneSeq
EP906954
Chemokines are a family of
Chemokine activities can be
Inflammatory, immune and


monocyte
Accession

related small, secreted proteins
determined using assays known in
infectious diseases; pulmonary


chemotactic
Y07234

involved in biological processes
the art: Methods in Molecular
diseases and skin disorders;


protein 2 (6-76)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
tumours, and angiogenesis-





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and haematopoiesis-related





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Truncated
GeneSeq
EP905241; EP906954
Chemokines are a family of
Chemokine activities can be
Inflammatory, immune and


RANTES
Accessions

related small, secreted proteins
determined using assays known in
infectious diseases; pulmonary


protein (3-68)
Y07236 and

involved in biological processes
the art: Methods in Molecular
diseases and skin disorders;



Y07232

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
tumours, and angiogenesis-





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and haematopoiesis-related





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Wild type
GeneSeq
EP905241
Chemokines are a family of
Chemokine activities can be
Inflammatory, immune and


monocyte
Accession

related small, secreted proteins
determined using assays known in
infectious diseases; pulmonary


chemotactic
Y07237

involved in biological processes
the art: Methods in Molecular
diseases and skin disorders;


protein 2


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
tumours, and angiogenesis-





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and haematopoiesis-related





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Truncated
GeneSeq
EP905241
Chemokines are a family of
Chemokine activities can be
Inflammatory, immune and


monocyte
Accession

related small, secreted proteins
determined using assays known in
infectious diseases; pulmonary


chemotactic
Y07238

involved in biological processes
the art: Methods in Molecular
diseases and skin disorders;


protein 2 (6-76)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
tumours, and angiogenesis-





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
and haematopoiesis-related





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


A partial
GeneSeq
EP897980
Chemokines are a family of
Chemokine activities can be
Soluble CXCR4B receptor


CXCR4B
Accession

related small, secreted proteins
determined using assays known in
polypeptides may be useful for


protein
W97363

involved in biological processes
the art: Methods in Molecular
inhibiting chemokine activities





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
and viral infection.





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Interferon
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


gamma-
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


inducible
W96709

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


protein (IP-10)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


A monokine
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


induced by
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


gamma-
W96710

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


interferon


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal


(MIG)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Interleukin-8
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


(IL-8) protein.
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune



W96711

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ; and Holmes et al





including inflammation, allergy,
(1991) Science 253, 1278-80.





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Epithelial
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


neutrophil
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


activating
W96712

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


protein-78


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal


(ENA-78)


angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Growth related
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


oncogene-alpha
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


(GRO-alpha).
W96713

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Growth related
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


oncogene-beta
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


(GRO-beta).
W96714

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Growth related
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


oncogene-
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


gamma (GRO-
W96715

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


gamma)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


A platelet basic
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


protein (PBP)
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune



W96716

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Connective
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


tissue activating
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


protein-III
W96717

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


(CTAP-III)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Beta-
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


thromboglobulin
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


protein (beta-
W96718

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


TG)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Neutrophil
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


activating
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


peptide-2
W96719

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


(NAP-2)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Granulocyte
GeneSeq
U.S. Pat. No. 5871723
Chemokines are a family of
Chemokine activities can be
Angiogenesis, Cancer,


chemotactic
Accession

related small, secreted proteins
determined using assays known in
Inflammatory and Immune


protein-2 (GCP-
W96720

involved in biological processes
the art: Methods in Molecular
disorders, Cardio-Vascular


2)


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
disorders, Musco-skeletal





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
disorders





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
EP887409
Chemokines are a family of
Chemokine activities can be
Immune disorders, viral,


chemokine
Accession

related small, secreted proteins
determined using assays known in
parasitic, fungal or bacterial


MIG-beta
W90124

involved in biological processes
the art: Methods in Molecular
infections, Cancer;


protein


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
autoimmune diseases or





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
transplant rejection





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.





family are involved in a similarly
Power.  © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO9854326
Chemokines are a family of
Chemokine activities can be
Immune disorders, cancer,


ZCHEMO-8
Accession

related small, secreted proteins
determined using assays known in
myelopoietic disorders,


protein
W82716

involved in biological processes
the art: Methods in Molecular
autoimmune disorders and





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
immunodeficiencies,





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
Inflammatory and infectious





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases, Vascular disorders,





family are involved in a similarly
Power.  © Humana Press Inc.,
wound healing





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human Act-2
GeneSeq
WO9854326
Chemokines are a family of
Chemokine activities can be
Immune disorders, cancer,


protein
Accession

related small, secreted proteins
determined using assays known in
myelopoietic disorders,



W82717

involved in biological processes
the art: Methods in Molecular
autoimmune disorders and





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
immunodeficiencies,





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
Inflammatory and infectious





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases, Vascular disorders,





family are involved in a similarly
Power.  © Humana Press Inc.,
wound healing





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human SISD
GeneSeq
WO9854326
Chemokines are a family of
Chemokine activities can be
Immune disorders, cancer,


protein
Accession

related small, secreted proteins
determined using assays known in
myelopoietic disorders,



W82720

involved in biological processes
the art: Methods in Molecular
autoimmune disorders and





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
immunodeficiencies,





angiogenesis, and leukocyte
Protocols. Edited by: A. E. I.
Inflammatory and infectious





trafficking. Members of this
Proudfoot, T. N. C. Wells, and C. A.
diseases, Vascular disorders,





family are involved in a similarly
Power.  © Humana Press Inc.,
wound healing





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human M110
GeneSeq
WO9854326
Chemokines are a family of
Chemokine activities can be
Immune disorders, cancer,


protein
Accession

related small, secreted proteins
determined using assays known in
myelopoietic disorders,



W82721

involved in biological processes
the art: Methods in Molecular
autoimmune disorders and





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
immunodeficiencies,





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.
Inflammatory and infectious





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.
diseases. Vascular disorders,





family are involved in a similarly
Power. © Humana Press Inc.,
wound healing





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





dcscribed, which bind to  ̃17





receptors thus far identified.


Human MIIA
GeneSeq
WO9854326
Chemokines are a family of
Chemokine activities can be
Immune disorders, cancer,


protein
Accession

related small, secreted proteins
determined using assays known in
myelopoietic disorders,



W82722

involved in biological processes
the art: Methods in Molecular
autoimmune disorders and





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
immunodeficiencies,





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.
Inflammatory and infectious





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.
diseases, Vascular disorders,





family are involved in a similarly
Power. © Humana Press Inc.,
wound healing





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human CCC3
GeneSeq
WO9854326
Chemokines are a family of
Chemokine activities can be
Immune disorders, cancer,


protein
Accession

related small, secreted proteins
determined using assays known in
myelopoietic disorders,



W82723

involved in biological processes
the art: Methods in Molecular
autoimmune disorders and





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
immunodeficiencies,





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.
Inflammatory and infectious





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.
diseases, Vascular disorders,





family are involved in a similarly
Power. © Humana Press Inc.,
wound healing





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


A human L105
GcneSeq
WO9856818
Chemokines are a family of
Chemokine activities can be
Cancer, wound healing,


chemokine
Accession

related small, secreted proteins
determined using assays known in


designated
W87588

involved in biological processes
the art: Methods in Molecular


huL105_3.


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


A human L105
GeneSeq
WO9856818
Chemokines are a family of
Chemokine activities can be
Cancer, wound healing,


chemokine
Accession

related small, secreted proteins
determined using assays known in


designated
W87589

involved in biological processes
the art: Methods in Molecular


huL105_7.


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Member of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptor. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human mature
GeneSeq
WO9848828
Chemokines are a family of
Chemokine activities can be
Infectious diseases, sepsis


gro-alpha
Accession

related small, secreted proteins
determined using assays known in


polypeptide
W81498

involved in biological processes
the art: Methods in Molecular


used to treat


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


sepsis


angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologics
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection.





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human mature
GeneSeq
WO9848828
Chemokines are a family of
Chemokine activities can be
Infectious diseases, sepsis


gro-gamma
Accession

related small, secreted proteins
determined using assays known in


polypeptide
W81500

involved in biological processes
the art: Methods in Molecular


used to treat


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


sepsis


angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human thymus
GeneSeq
WO0053635
Chemokines are a family of
Chemokine activities can be
Inflammatory disorders,


expressed
Accessions

related small secreted proteins
determined using assays known in
cancer, Immune and vascular


chemokine
B19607 and

involved in biological processes
the art: Methods in Molecular
disorders


TECK and
B19608

ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


TECK variant


angiogenesis and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15791

related small, secreted proteins
determined using assays known in
Immune, Vascular and


SDF1alpha


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15793

related small, secreted proteins
determined using assays known in
Immune, Vascular and


GROalpha


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15794

related small, secreted proteins
determined using assays known in
Immune, Vascular and


cotaxin


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine MIG
Accession B15803

related small, secreted proteins
determined using assays known in
Immune, Vascular and





involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine PF4
Accession B15804

related small, secreted proteins
determined using assays known in
Immune, Vascular and





involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine 1-
Accession B15805

related small, secreted proteins
determined using assays known in
Immune, Vascular and


309


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15806

related small, secreted proteins
determined using assays known in
Immune, Vascular and


HCC-1


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine C10
Accession B15807

related small, secreted proteins
determined using assays known in
Immune, Vascular and





involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15808

related small, secreted proteins
determined using assays known in
Immune, Vascular and


CCR-2


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15809

related small, secreted proteins
determined using assays known in
Immune, Vascular and


ENA-78


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15810

related small, secreted proteins
determined using assays known in
Immune, Vascular and


GRObeta


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine IP-
Accession B15811

related small, secreted proteins
determined using assays known in
Immune, Vascular and


10


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15812

related small, secreted proteins
determined using assays known in
Immune, Vascular and


SDF1beta


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15813

related small, secreted proteins
determined using assays known in
Immune, Vascular and


GRO alpha


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0042071
Chemokines are a family of
Chemokine activities can be
Autoimmune disorders,


chemokine
Accession B15831

related small, secreted proteins
determined using assays known in
Immune, Vascular and


MIP1beta


involved in biological processes
the art: Methods in Molecular
Inflammatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


A human C-C
GeneSeq
U.S. Pat. No. 6096300
Chemokines are a family of
Chemokine activities can be
Cancer


chemokine
Accession B07939

related small, secreted proteins
determined using assays known in


designated


involved in biological processes
the art: Methods in Molecular


exodus


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
U.S. Pat. No. 6084071
Chemokines are a family of
Chemokine activities can be
Chemotaxis, Gene Therapy,


chemokine
Accession

related small, secreted proteins
determined using assays known in
Wound healing


L105_7
Y96922

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
U.S. Pat. No. 6084071
Chemokines are a family of
Chemokine activities can be
Chemotaxis, Gene Therapy,


chemokine
Accession

related small, secreted proteins
determined using assays known in
Wound healing


L105_3
Y96923

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0038706
Chemokines are a family of
Chemokine activities can be
Cancer, Vascular and Immune


secondary
Accession B01434

related small, secreted proteins
determined using assays known in
disorders


lymphoid


involved in biological processes
the art: Methods in Molecular


chemokine


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


(SLC)


angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human non-
GeneSeq
WO0029439
Chemokines are a family of
Chemokine activities can be
Immune and Inflammatory


ELR CXC
Accession

related small, secreted proteins
determined using assays known in
disorders, Cancer, haemostatic


chemokine
Y96310

involved in biological processes
the art: Methods in Molecular
and thrombolytic activity


H174.


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human non-
GeneSeq
WO0029439
Chemokines are a family of
Chemokine activities can be
Immune and Inflammatory


ELR CXC
Accession

related small, secreted proteins
determined using assays known in
disorders, Cancer, haemostatic


chemokine IP10
Y96311

involved in biological processes
the art: Methods in Molecular
and thrombolytic activity





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human non-
GeneSeq
WO0029439
Chemokines are a family of
Chemokine activities can be
Immune and Inflammatory


ELR CXC
Accession

related small, secreted proteins
determined using assays known in
disorders, Cancer, haemostatic


chemokine Mig
Y96313

involved in biological processes
the art: Methods in Molecular
and thrombolytic activity





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0028035
Chemokines are a family of
Chemokine activities can be
Cancer, wound healing,


chemokine
Accession

related small, secreted proteins
determined using assays known in
inflammatory and


Ckbeta-7
Y96280

involved in biological processes
the art: Methods in Molecular
immunoregulatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0028035
Chemokines are a family of
Chemokine activities can be
Cancer, wound healing,


chemokine
Accession

related small, secreted proteins
determined using assays known in
inflammatory and


MIP-1alpha
Y96281

involved in biological processes
the art: Methods in Molecular
immunoregulatory disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human mature
GeneSeq
WO0028035
Chemokines are a family of
Chemokine activities can be
Cancer, wound healing,


chemokine
Accession

related small, secreted proteins
determined using assays known in
inflammatory and


Ckbeta-7
Y96282

involved in biological processes
the art: Methods in Molecular
immunoregulatory disorders


(optionally


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine


truncated)


angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
WO0018431
Chemokines are a family of
Chemokine activities can be
Soluble CXCR3 polypeptides


chemokine
Accession

related small, secreted proteins
determined using assays known in
may be useful for inhibiting


receptor
Y79372

involved in biological processes
the art: Methods in Molecular
chemokine activities and viral


CXCR3


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine
infection.





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
U.S. Pat. No. 6043086
Chemokines are a family of
Chemokine activities can be
Neurological disorders,


neurotactin
Accession

related small, secreted proteins
determined using assays known in
Immune and respiratory


chemokine like
Y53259

involved in biological processes
the art: Methods in Molecular
disorders


domain


ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human CC type
GeneSeq
JP11302298
Chemokines are a family of
Chemokine activities can be
Cancer and infectious diseases


chemokine
Accession

related small, secreted proteins
determined using assays known in


interleukin C
Y57771

involved in biological processes
the art: Methods in Molecular





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Human
GeneSeq
U.S. Pat. No. 6153441
Chemokines are a family of
Chemokine activities can be
Cancer, Auto-immune and


CKbeta-9
Accession

related small, secreted proteins
determined using assays known in
inflammatory disorders,



B50860

involved in biological processes
the art: Methods in Molecular
Cardiovasular disorders





ranging from hematopoiesis,
Biology, 2000, vol. 138: Chemokine





angiogenesis, and leukocyte
Protocols. Edited by: A.E.I.





trafficking. Members of this
Proudfoot, T.N.C. Wells, and C.A.





family are involved in a similarly
Power. © Humana Press Inc.,





diverse range of pathologies
Totowa, NJ





including inflammation, allergy,





tissue rejection, viral infection,





and tumor biology. The





chemokines exert their effects by





acting on a family of seven





transmembrane G-protein-





coupled receptors. Over 40





human chemokines have been





described, which bind to  ̃17





receptors thus far identified.


Preproapolipoprotein
GeneSeq
WO9637608
apoa-1 participates in the reverse
Lipid binding activity can be
Useful for cardiovascular


“paris”
Accession

transport of cholesterol from
determined using assays known in
disorders, cholesterol


variant
W08602

tissues to the liver for excretion
the art, such as, for example, the
disorders, and





by promoting cholesterol efflux
Cholesterol Efflux Assays of
Hyperlipidaemia





from tissues and by acting as a
Takahashi et al., P.N.A.S., Vol. 96,





cofactor for the lecithin
issue 20, 11358-11363, Sept.





cholesterol acyltransferase
28, 1999.





(lcat).


Preproapolipoprotein

5,721,114
apoa-1 participates in the reverse
Lipid binding activity can be
Useful for cardiovascular


“milano”


transport of cholesterol from
determined using assays known in
disorders, cholesterol


variant


tissues to the liver for excretion
the art, such as, for example, the
disorders, and





by promoting cholesterol efflux
Cholesterol Efflux Assays of
Hyperlipidaemia





from tissues and by acting as a
Takabashi et al., P.N.A.S., Vol. 96,





cofactor for the lecithin
issue 20, 11358-11363, Sept.





cholesterol acyltransferase
28, 1999.





(lcat).


Glycodelin-A;
GeneSeq
WO9628169
Naturally produced female
Glycodelin-A activity can be
Naturally derived


Progesterone-
Accession

contraceptive that is removed
determined using the hemizona
contraceptive useful for the


associated
W00289

rapidly from the body following
assay as described in Oehninger, S.,
prevention of pregnancy.


endometrial


2-3 days production. Uses
Coddington, C. C., Hodgen, G. D.,


protein


include contraception
and Seppala. M. (1995) Fertil. Steril.






63, 377-383.


NOGO-A
Genbank

NOGO polypeptides are potent
Inhibition of Neurite outgrowth.
NOGO-A polypeptide



Accession

inhibitors of neurite growth.
Antagonists to NOGO polypeptides
antagonists are useful for the



CAB99248


may promote the outgrowth of
promotion of neural growth,






neurites, thus inducing regeneration
which could be useful in the






of neurons.
treatment of neural disorders







and dysfunction due to







degenerative diseases or







trauma; useful in the treatment







of neoplastic diseases of the







CNS; induce regeneration of







neurons or to promote the







structural plasticity of the







CNS.


NOGO-B
Genbank

NOGO polypeptides are potent
Inhibition of Neurite outgrowth.
NOGO-B polypeptide



Accession

inhibitors of neurite growth.
Antagonists to NOGO polypeptides
antagonists are useful for the



CAB99249


may promote the outgrowth of
promotion of neural growth,






neurites, thus inducing regeneration
which could be useful in the






of neurons.
treatment of neural disorders







and dysfunction due to







degenerative diseases or







trauma; useful in the treatment







of neoplastic diseases of the







CNS; induce regeneration of







neurons or to promote the







structural plasticity of the







CNS.


NOGO-C
Genbank

NOGO polypeptides are potent
Inhibition of Neurite outgrowth.
NOGO-C polypeptide



Accession

inhibitors of neurite growth.
Antagonists to NOGO polypeptides
antagonists are useful for the



CAB99250


may promote the outgrowth of
promotion of neural growth,






neurites, thus inducing regeneration
which could be useful in the






of neurons.
treatment of neural disorders







and dysfunction due to







degenerative diseases or







trauma; useful in the treatment







of neoplastic diseases of the







CNS; induce regeneration of







neurons or to promote the







structural plasticity of the







CNS.


NOGO-66
Genbank

NOGO polypeptides are potent
Inhibition of Neurite outgrowth.
Soluble NOGO-66 receptor


Receptor
Accession

inhibitors of neurite growth, and
mediating the biologicl effects of
polypeptides are useful for the



AAG53612

are thought to mediate their
NOGO polypeptides. Soluble
promotion of neural growth,





effects through the NOGO-66
NOGO-66 receptor polypeptides
which could be useful in the





Receptor.
may promote the outgrowth of
treatment of neural disorders






neurities, thus inducing regeneration
and dysfunction due to






of neurons.
degenerative diseases or







trauma; useful in the treatment







of neoplastic diseases of the







CNS; induce regeneration of







neurons or to promote the







structural plasticity of the







CNS.


Antibodies

U.S. Pat. No. 5416197
These antibodies are useful for
Collapsin activity, which is thought
Useful for the promotion of


specific for


the promotion of neurite
to inhibit the outgrowth of neurites,
neural growth, which could be


collapsin


outgrowth
can be assayed in the presence of
useful in the treatment of






antibodies specific for collapsing
neural disorders and






using assays known in the art, such
dysfunction due to






as, for example, the collapse assay
degenerative diseases or






disclosed by Luo et al., Cell 1993
trauma.






Oct. 22; 75(2):217-27.


Humanized

WO9845331
These agents have anti-
VEGF activity can be determined
Promotion of growth and


Anti-VEGF


inflammatory and anti-cancer
using assays known in the art, such
proliferation of cells, such as


Antibodies, and


applications
as those disclosed in International
vascular endothelial cells.


fragments



Publication No. WO0045835, for
Antagonisis may be useful as


thereof



example.
anti-angiogenic agents, and







may be applicable for cancer


Humanized

WO0029584
These agents have anti-
VEGF activity can be determined
Promotion of growth and


Anti-VEGF


inflammatory and anti-cancer
using assays known in the art, such
proliferation of cells, such as


Antibodies, and


applications
as those disclosed in International
vascular endothelial cells.


fragments



Publication No. WO0045835, for
Antagonists may be useful as


thereof



example.
anti-angiogenic agents, and







may be applicable for cancer


Membrane-
GeneSeq
WO9963088
Cancer, Immune Disorders
These proteins can be used for
Activities can be determined


bound proteins
Accession


linking bioactive molecules to cells
using assay known in the art,



Y66631-Y66765


and for modulating biological
such as, for example, the assays






activities of cells, using the
disclosed in International






polypeptides for specific targeting.
Publication No. WO0121658.






The polypeptide targeting can be






used to kill the target cells, e.g. for






the treatment of cancers. These






proteins are useful for the treatment






of immune system disorders.


Secreted and
GeneSeq
WO0053756
Cancer, Immune Disorders
These proteins can be used for
Activities can be determined


Transmembrane
Accession


linking bioactive molecules to cells
using assay known in the art,


polypeptides
B44241-B44334


and for modulating biological
such as, for example, the assays






activities of cells, using the
disclosed in international






polypeptides for specific targeting.
Publication No. WO0121658.






The polypeptide targeting can be






used to kill the target cells, e.g. for






the treatment of cancers. These






proteins are useful for the treatment






of immune system disorders.


Secreted and
GeneSeq
WO9946281
Cancer, Immune Disorders
These proteins can be used for
Activities can be determined


Transmembrane
Accession


linking bioactive molecules to cells
using assay known in the art,


polypeptides
Y41685-Y41774


and for modulating biological
such as, for example, the assays






activities of cells, using the
disclosed in International






polypeptides for specific targeting.
Publication No. WO0121658.






The polypeptide targeting can be






used to kill the target cells, e.g. for






the treatment of cancers. These






proteins are useful for the treatment






of immune system disorders.









In preferred embodiments, the albumin fusion proteins of the invention are capable of a therapeutic activity and/or biologic activity corresponding to the therapeutic activity and/or biologic activity of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein listed in the corresponding row of Table 1. (See, e.g., the “Biological Activity” and “Therapeutic Protein X”columns of Table 1.) In further preferred embodiments, the therapeutically active protein portions of the albumin fusion proteins of the invention are fragments or variants of the reference sequence cited in the “Exemplary Identifier” column of Table 1, and are capable of the therapeutic activity and/or biologic activity of the corresponding Therapeutic protein disclosed in “Biological Activity” column of Table 1.
Polypeptide and Polynucleotide Fragments and Variants
Fragments
The present invention is further directed to fragments of the Therapeutic proteins described in Table 1, albumin proteins, and/or albumin fusion proteins of the invention.
Even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the Therapeutic protein, albumin protein, and/or albumin fusion protein, other Therapeutic activities and/or functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of polypeptides with N-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
Accordingly, fragments of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (e.g., a Therapeutic protein as disclosed in Table 1). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, fragments of serum albumin polypeptides corresponding to an albumin protein portion of an albumin fusion protein of the invention, include the full length protein as well as polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the reference polypeptide (i.e., serum albumin). In particular, N-terminal deletions may be described by the general formula m-585, where 585 is a whole integer representing the total number of amino acid residues in serum albumin (SEQ ID NO: 18), and m is defined as any integer ranging from 2 to 579. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Moreover, fragments of albumin fusion proteins of the invention, include the full length albumin fusion protein as well as polypeptides having one or more residues deleted from the amino terminus of the albumin fusion protein. In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in the albumin fusion protein, and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the N-terminus or C-terminus of a reference polypeptide (e.g., a Therapeutic protein and/or serum albumin protein) results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) and/or Therapeutic activities may still be retained. For example the ability of polypeptides with C-terminal deletions to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking the N-terminal and/or C-terminal residues of a reference polypeptide retains Therapeutic activity can readily be determined by routine methods described herein and/or otherwise known in the art.
The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., a Therapeutic protein referred to in Table 1). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where q is a whole integer representing the total number of amino acid residues in a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1). Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention (e.g., serum albumin). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to 584, where 584 is the whole integer representing the total number of amino acid residues in serum albumin (SEQ ID NO:18) minus 1. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Moreover, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus of an albumin fusion protein of the invention. In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where q is a whole integer representing the total number of amino acid residues in an albumin fusion protein of the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted reference polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a reference polypeptide (e.g., a Therapeutic protein referred to in Table 1, or serum albumin (e.g., SEQ ID NO:18), or an albumin fusion protein of the invention) where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference polypeptide sequence (e.g., a Therapeutic protein, serum albumin protein or an albumin fusion protein of the invention) set forth herein, or fragments thereof. In preferred embodiments, the application is directed to proteins comprising polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to reference polypeptides having the amino acid sequence of N- and C-terminal deletions as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a Therapeutic activity and/or functional activity (e.g. biological activity) of the polypeptide sequence of the Therapeutic protein or serum albumin protein of which the amino acid sequence is a fragment
Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Variants
“Variant” refers to a polynucleotide or nucleic acid differing from a reference nucleic acid or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the reference nucleic acid or polypeptide.
As used herein, “variant”, refers to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin portion of an albumin fusion protein of the invention, or albumin fusion protein differing in sequence from a Therapeutic protein (e.g. see “therapeutic” column of Table 1), albumin protein, and/or albumin fusion protein of the invention, respectively, but retaining at least one functional and/or therapeutic property thereof (e.g., a therapeutic activity and/or biological activity as disclosed in the “Biological Activity” column of Table 1) as described elsewhere herein or otherwise known in the art. Generally, variants are overall very similar, and, in many regions, identical to the amino acid sequence of the Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention, albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention, and/or albumin fusion protein of the invention. Nucleic acids encoding these variants are also encompassed by the invention.
The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, the amino acid sequence of a Therapeutic protein corresponding to a Therapeutic protein portion of an albumin fusion protein of the invention (e.g., an amino acid sequence disclosed in the “Exemplary Identifier” column of Table 1, or fragments or variants thereof), albumin proteins (e.g., SEQ ID NO:18 or fragments or variants thereof) corresponding to an albumin protein portion of an albumin fusion protein of the invention, and/or albumin fusion proteins of the invention. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding an amino acid sequence of the invention under stringent hybridization conditions (e.g., hybridization to filter bound DNA in 6×Sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2×SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (e.g., hybridization to filter bound DNA in 6×sodium chloride/Sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.1×SSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989 Current protocol in Molecular Biology, Green publishing associates, Inc., and John Wiley & Sons. Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3). Polynucleotides encoding these polypeptides are also encompassed by the invention.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 917%, 98% or 99% identical to, for instance, the amino acid sequence of an albumin fusion protein of the invention or a fragment thereof (such as the Therapeutic protein portion of the albumin fusion protein or the albumin portion of the albumin fusion protein), can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
The variant will usually have at least 75% (preferably at least about 80%, 90%, 95% or 99%) sequence identity with a length of normal HA or Therapeutic protein which is the same length as the variant. Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et at, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36: 290-300 (1993), fully incorporated by reference) which are tailored for sequence similarity searching.
The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al., (Nature Genetics 6: 119-129 (1994)) which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated by reference). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and −4, respectively. Four blasm parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host, such as, yeast or E. coli).
In a preferred embodiment, a polynucleotide encoding an albumin portion of an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells. In further preferred embodiment, a polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells. In a still further preferred embodiment, a polynucleotide encoding an albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.
In an alternative embodiment, a codon optimized polynucleotide encoding a Therapeutic protein portion of an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the Therapeutic protein under stringent hybridization conditions as described herein. In a further embodiment, a codon optimized polynucleotide encoding an albumin portion of an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the albumin protein under stringent hybridization conditions as described herein. In another embodiment, a codon optimized polynucleotide encoding an albumin fusion protein of the invention does not hybridize to the wild type polynucleotide encoding the Therapeutic protein portin or the albumin protein portion under stringent hybridization conditions as described herein.
In an additional embodiment, polynucleotides encoding a Therapeutic protein portion of an albumin fusion protein of the invention do not comprise, or alternatively consist of, the naturally occurring sequence of that Therapeutic protein. In a further embodiment, polynucleotides encoding an albumin protein portion of an albumin fusion protein of the invention do not comprise, or alternatively consist of, the naturally occurring sequence of albumin protein. In an alternative embodiment, polynucleotides encoding an albumin fusion protein of the invention do not comprise, or alternatively consist of, of the naturally occurring sequence of a Therapeutic protein portion or the albumin protein portion.
Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Thus, the invention further includes polypeptide variants which have a functional activity (e.g., biological activity and/or therapeutic activity). In highly preferred embodiments the invention provides variants of albumin fusion proteins that have a functional activity (e.g., biological activity and/or therapeutic activity, such as that disclosed in the “Biological Activity” column in Table 1) that corresponds to one or more biological and/or therapeutic activities of the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
In preferred embodiments, the variants of the invention have conservative substitutions. By “conservative substitutions” is intended swaps within groups such as replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr, replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Guidance concerning how to make phenotypically silent amino acid substitutions is provided, for example, in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) polypeptides containing substitutions of one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) polypeptides containing substitutions of one or more of the amino acid residues having a substituent group, or (iii) polypeptides which have been fused with or chemically conjugated to another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) polypeptide containing additional amino acids, such as, for example, an IgG Fc fusion region peptide, Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of the amino acid sequence of a Therapeutic protein described herein and/or human serum albumin, and/or albumin fusion protein of the invention, wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the invention.
The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttransiation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Functional Activity
“A polypeptide having functional activity” refers to a polypeptide capable of displaying one or more known functional activities associated with the full-length, pro-protein, and/or mature form of a Therapeutic protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
“A polypeptide having biological activity” refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a Therapeutic protein of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit oreater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
In preferred embodiments, an albumin fusion protein of the invention has at least one biological and/or therapeutic activity associated with the Therapeutic protein (or fragment or variant thereof) when it is not fused to albumin.
The albumin fusion proteins of the invention can be assayed for functional activity (e.g., biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, albumin fusion proteins may be assayed for functional activity (e.g., biological activity or therapeutic activity) using the assay referenced in the “Exemplary Activity Assay” column of Table 1. Additionally, one of skill in the art may routinely assay fragments of a Therapeutic protein corresponding to a. Therapeutic protein portion of an albumin fusion protein of the invention, for activity using assays referenced in its corresponding row of Table 1. Further, one of skill in the art may routinely assay fragments of an albumin protein corresponding to an albumin protein portion of an albumin fusion protein of the invention, for activity using assays known in the art and/or as described in the Examples section below.
For example, in one embodiment where one is assaying for the ability of an albumin fusion protein of the invention to bind or compete with a Therapeutic protein for binding to an anti-Therapeutic polypeptide antibody and/or anti-albumin antibody, various immunoassays known in the art can be used, including but not limited to, competitive and noncompetitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemaggluinarion assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In a preferred embodiment, where a binding partner (e.g., a receptor or a ligand) of a Therapeutic protein is identified, binding to that binding partner by an albumin fusion protein containing that Therapeutic protein as the Therapeutic protein portion of the fusion can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of an albumin fusion protein of the present invention to bind to a substrate(s) of the Therapeutic polypeptide corresponding to the Therapeutic portion of the albumin fusion protein of the invention can be routinely assayed using techniques known in the art.
In an alternative embodiment, where the ability of an albumin fusion protein of the invention to multimerize is being evaluated, association with other components of the multimer can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., supra.
In preferred embodiments, an albumin fusion protein of the invention comprising all or a portion of an antibody that binds a Therapeutic protein, has at least one biological and/or therapeutic activity (e.g., to specifically bind a polypeptide or epitope) associated with the antibody that binds a Therapeutic protein (or fragment or variant thereof) when it is not fused to albumin. In other preferred embodiments, the biological activity and/or therapeutic activity of an albumin fusion protein of the invention comprising all or a portion of an antibody that binds a Therapeutic protein is the inhibition (i.e. antagonism) or activation (i.e., agonism) of one or more of the biological activities and/or therapeutic activities associated with the polypeptide that is specifically bound by antibody that binds a Therapeutic protein.
Albumin fusion proteins of the invention (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) may be characterized in a variety of ways. In particular, albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for the ability to specifically bind to the same antigens specifically bound by the antibody that binds a Therapeutic protein corresponding to the Therapeutic protein portion of the albumin fusion protein using techniques described herein or routinely modifying techniques known in the art.
Assays for the ability of the albumin fusion proteins of the invention (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to (specifically) bind a specific protein or epitope may be performed in solution (e.g., Houghten, Bio/Techniques 13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)) (each of these references is incorporated herein in its entirety by reference). Albumin fusion proteins of the invention comprising at least a fragment or variant of a Therapeutic antibody may also be assayed for their specificity and affinity for a specific protein or epitope using or routinely modifying techniques described herein or otherwise known in the art.
The albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for cross-reactivity with other antigens (e.g., molecules that have sequence/structure conservation with the molecule(s) specifically bound by the antibody that binds a Therapeutic protein (or fragment or variant thereof) corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention) by any method known in the art.
Immunoassays which can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the albumin fusion protein of the invention (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C., adding sepharose beads coupled to an anti-albumin antibody, for example, to the cell lysate, incubating for about an hour or more at 40 degrees C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the albumin fusion protein of the invention to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the albumin fusion protein to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), applying the albumin fusion protein of the invention (diluted in blocking buffer) to the membrane, washing the membrane in washing buffer, applying a secondary antibody (which recognizes the albumin fusion protein, e.g., an anti-human serum albumin antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the albumin fusion protein (e.g., comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) of the invention conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound or non-specifically bound albumin fusion proteins, and detecting the presence of the albumin fusion proteins specifically bound to the antigen coating the well. In ELISAs the albumin fusion protein does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes albumin fusion protein) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the albumin fusion protein may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an albumin fusion protein to a protein, antigen, or epitope and the off-rate of an albumin fusion protein-protein/antigen/epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the albumin fusion protein of the present invention for a specific protein, antigen, or epitope and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second protein that binds the same protein, antigen or epitope as the albumin fusion protein, can also be determined using radioimmunoassays. In this case, the protein, antigen or epitope is incubated with an albumin fusion protein of the present invention conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second protein that binds the same protein, antigen, or epitope as the albumin fusion protein of the invention.
In a preferred embodiment, BLAcore kinetic analysis is used to determine the binding on and off rates of albumin fusion proteins of the invention to a protein, antigen or epitope. Acore kinetic analysis comprises analyzing the binding and dissociation of albumin fusion proteins, or specific polypeptides, antigens or epitopes from chips with immobilized specific polypeptides, antigens or epitopes or albumin fusion proteins, respectively, on their surface.
Antibodies that bind a Therapeutic protein corresponding to the Therapeutic protein portion of an albumin fusion protein of the invention may also be described or specified in terms of their binding affinity for a given protein or antigen, preferably the antigen which they specifically bind. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 binding affinities include those with a dissociation constant or Kd less than 5×10−5 M, 10−5 M, 5×10−6 M, 10−6M, 5×10−7 M, 10−7 M, 5×10−8 M or 10−8 M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10-12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has an affinity for a given protein or epitope similar to that of the corresponding antibody (not fused to albumin) that binds a Therapeutic protein, taking into account the valency of the albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and the valency of the corresponding antibody.
In addition, assays described herein (see Examples and Table 1) and otherwise known in the art may routinely be applied to measure the ability of albumin fusion proteins of the present invention and fragments, variants and derivatives thereof to elicit biological activity and/or Therapeutic activity (either in vitro or in vivo) related to either the Therapeutic protein portion and/or albumin portion of the albumin fusion protein of the present invention. Other methods will be known to the skilled artisan and are within the scope of the invention.
Albumin
As described above, an albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion or chemical, conjugation.
The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms, “albumin and “serum albumin” are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
As used herein, “albumin” refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, “albumin” refers to human albumin or fragments thereof (see EP 201 239, EP 322 094 WO 97/24445, WO95/23857) especially the mature form of human albumin as shown in FIG. 15 and SEQ ID NO:18, or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.
In preferred embodiments, the human serum albumin protein used in the albumin fusion proteins of the invention contains one or both of the following sets of point mutations with reference to SEQ ID NO: 18: Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to A; Lys-413 to Gin, and Lys-414 to Gln (see, e.g., International Publication No. WO95/23857, hereby incorporated in its entirety by reference herein). In even more preferred embodiments, albumin fusion proteins of the invention that contain one or both of above-described sets of point mutations have improved stability/resistance to yeast Yap3p proteolytic cleavage, allowing increased production of recombinant albumin fusion proteins expressed in yeast host cells.
As used herein, a portion of albumin sufficient to prolong the therapeutic activity or shelf-life of the Therapeutic protein refers to a portion of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity of the protein so that the shelf life of the Therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the shelf-life in the non-fusion state. The albumin portion of the albumin fusion proteins may comprise the full length of the HA sequence as described above or as shown in FIG. 15, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity. Such fragments may be of 10 or more amino acids in length or may include about 15, 20,25, 30, 50, or more contiguous amino acids from the HA sequence or may include part or all of specific domains of HA. For instance, one or more fragments of HA spanning the first two immunoglobulin-like domains may be used.
The albumin portion of the albumin fusion proteins of the invention may be a variant of normal HA. The Therapeutic protein portion of the albumin fusion proteins of the invention may also be variants of the Therapeutic proteins as described herein. The term “variants” includes insertions, deletions and substitutions, either conservative or non conservative, where such changes do not substantially alter one or more of the oncotic, useful ligand-binding and non-immunogenic properties of albumin, or the active site, or active domain which confers the therapeutic activities of the Therapeutic proteins.
In particular, the albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin, for example those fragments disclosed in EP 322 094 (namely HA (Pn), where n is 369 to 419). The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the Therapeutic protein portion.
Generally speaking, an HA fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long. The HA variant may consist of or alternatively comprise at least one whole domain of HA, for example domains 1 (amino acids 1-194 of SEQ ID NO: 18), 2 (amino acids 195-387 of SEQ ID NO: 18), 3 (amino acids 388-585 of SEQ ID NO:18), 1+2 (1-387 of SEQ ID NO: 18), 2+3 (195-585 of SEQ ID NO: 18 (amino acids 1-194 of SEQ ID NO: 18+amino acids 388-585 of SEQ ID NO:18). Each domain is itself made up of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to Glu119, Glu292 to Val315 and Glu492 to Ala511.
Preferably, the albumin portion of an albumin fusion protein of the invention comprises at least one subdomain or domain of HA or conservative modifications thereof. If the fusion is based on subdomains, some or all of the adjacent linker is preferably used to link to the Therapeutic protein moiety.
Antibodies that Specifically Bind Therapeutic Proteins are also Therapeutic Proteins
The present invention also encompasses albumin fusion proteins that comprise at least a fragment or variant of an antibody that specifically binds a Therapeutic protein disclosed in Table 1. It is specifically contemplated that the term “Therapeutic protein” encompasses antibodies that bind a Therapeutic protein and fragments and variants thereof. Thus an albumin fusion protein of the invention may contain at least a fragment or variant of a Therapeutic protein, and/or at least a fragment or variant of an an antibody that binds a Therapeutic protein.
Antibody Structure and Background
The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “fight” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Chapters 3-5 (Paul, W., ed., 4th ed. Raven Press, N.Y. (1998)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.
Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.
The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDR regions, in general, are the portions of the antibody which make contact with the antigen and determine its specificity. The CDRs from the heavy and the light chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains variable regions comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions are connected to the heavy or light chain constant region. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
As used herein, “antibody” refers to immunoglobulin molecules and inmnunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen (e.g., a molecule containing one or more CDR regions of an antibody). Antibodies that may correspond to a Therapeutic protein portion of an albumin fusion protein include, but are not limited to, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies (e.g., single chain Fvs), Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library; anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies specific to antibodies of the invention), and epitope-binding fragments of any of the above (e.g., VH domains, VL domains, or one or more CDR regions).
Antibodies that Bind Therapeutic Proteins
The present invention encompasses albumin fusion proteins that comprise at least a fragment or variant of an antibody that binds a Therapeutic Protein (e.g., as disclosed in Table 1) or fragment or variant thereof.
Antibodies that bind a Therapeutic protein (or fragment or variant thereof) may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibodies. Most preferably, the antibodies are human antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries and xenomice or other organisms that have been genetically engineered to produce human antibodies.
The antibody molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the antibody molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention are IgG1. In other preferred embodiments, the immunoglobulin molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention are IgG2. In other preferred embodiments, the immunoglobulin molecules that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention are IgG4.
Most preferably the antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, singlehain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains.
The antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a Therapeutic protein or may be specific for both a Therapeutic protein as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., I. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies that bind a Therapeutic protein (or fragment or variant thereof) may be bispecific or bifunctional which means that the antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148:1547-1553 (1992). In addition, bispecific antibodies may be formed as “diabodies” (Holliger et al. “Diabodies': small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448 (1993)) or “Janusins” (Traunecker et al. “Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells” EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new molecular design for bispecific reagents” Int J Cancer Suppl 7:51-52 (1992)).
The present invention also provides albumin fusion proteins that comprise, fragments or variants (including derivatives) of an antibody described herein or known elsewhere in the art. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid subsitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. In specific embodiments, the variants encode substitutions of VHCDR3. In a preferred embodiment, the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues.
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may be described or specified in terms of the epitope(s) or portion(s) of a Therapeutic protein which they recognize or specifically bind. Antibodies which specifically bind a Therapeutic protein or a specific epitope of a Therapeutic protein may also be excluded. Therefore, the present invention encompasses antibodies that specifically bind Therapeutic proteins, and allows for the exclusion of the same. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, binds the same epitopes as the corresponding antibody (not fused to albumin) that binds a Therapeutic protein.
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a Therapeutic protein are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a Therapeutic protein are also included in the present invention. In specific embodiments, antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a Therapeutic protein are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has similar or substantially identical cross reactivity characteristics compared to the corresponding antibody (not fused to albumin) that binds a Therapeutic protein.
Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide encoding a Therapeutic protein under stringent hybridization conditions (as described herein). Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M. More preferred binding affinities include those with a dissociation constant or Kd less than 5×10−5 M, 10−5 M, 5×10−6 M, 106M, 5×10−7 M, 10−7 M, 5×10−8 M or 10−8 M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10-12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has an affinity for a given protein or epitope similar to that of the corresponding antibody (not fused to albumin) that binds a Therapeutic protein, taking into account the valency of the albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) and the valency of the corresponding antibody.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of a Therapeutic protein as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, competitively inhibits binding of an antibody to an epitope of a Therapeutic protein as well as the corresponding antibody (not fused to albumin) that binds a Therapeutic protein, competitively inhibits binding of an antibody to an epitope of a Therapeutic protein. In other preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, competitively inhibits binding of the corresponding antibody (not fused to albumin) that binds a Therapeutic protein to an epitope of a Therapeutic protein by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may act as agonists or antagonists of the Therapeutic protein. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphocrylation (e.g., tyrosine or serine/threonine)of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 800%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody. In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, has similar or substantially similar characteristics with regard to preventing ligand binding and/or preventing receptor activation compared to the corresponding antibody (not fused to albumin) that binds a Therapeutic protein.
The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the uhbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the Therapeutic protreins (e.g. as disclosed in Table 1). The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 178&1.794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):1420 (1996) (which are all incorporated by reference herein in their entireties). In preferred embodiments, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, have similar or substantially identical agonist or antagonist properties as the corresponding antibody that binds a Therapeutic protein not fused to albumin.
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention may be used, for example, to purify, detect, and target Therapeutic proteins, including both in in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the Therapeutic protein in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed 1988); incorporated by reference herein in its entirety. Likewise, albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, may be used, for example, to purify, detect, and target Therapeutic proteins, including both in in vitro and in vivo diagnostic and therapeutic methods.
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by lycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. Albumin fusion proteins of the invention may also be modified as described above.
Methods of Producing Antibodies that Bind Therapeutic Proteins
The antibodies that bind to a Therapeutic protein and that may correspond to, a Therapeutic protein portion of an albumin fusion protein of the invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a Therapeutic protein may be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.; 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with a Therapeutic protein or fragment or variant thereof or a cell expressing such a Therapeutic protein or fragment or variant thereof. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines maybe subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g. SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SK-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
For example, antibodies that bind to a Therapeutic protein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make antibodies that bind to a Therapeutic protein include those disclosed in Brinkman et at., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen an be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody and fragments thereof. The invention also encompase polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a Therapeutic protein, preferably, an antibody that binds to a polypeptide having the amino acid sequence of a “Therapeutic Protein X” as discosed in the “Exemplary Identifier” column of Table 1.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, ora cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art (see, Example 60).
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds. 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-419 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli many also be used (Skerra et al., Science 242:1038-1041 (1988)).
Recombinant Expression of Antibodies
Recombinant expression of an antibody, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody or a single chain antibody), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire imnmunoglobulin molecule, as detailed below.
A variety of hostexpression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santeire et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody genes production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutanaine synthase negative. Glutamine synthase expression systems can also function in glutanine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suppliers, including, for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entirities by reference herein.
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chainderived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention or fragments thereof can be fused to he terologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
Modifications of Antibodies
Antibodies that bind a Therapeutic protein or fragments or variants can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidinepeptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotn and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc. Other examples of detectable substances have been described elsewwhere herein.
Further, an antibody of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alphacmiters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiarnine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interieukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety. An antibody, with or without a therapeutic moiety conjugated to it administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
Antibody-albumin Fusion
Antibodies that bind to a Therapeutic protein and that may correspond to a Therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, antibodies that bind a Therapeutic protein disclosed in the “Therapeutic Protein X” column of Table 1, or a fragment or variant thereof.
In specific embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH domain. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, one, two or three VH CDRs. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH CDR1. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH CDR2. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VH CDR3.
In specific embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL domain. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, one, two or three VL CDRs. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL CDR1. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL CDR2. In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, the VL CDR3.
In other embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, one, two, three, four, five, or six VH and/or VL CDRs.
In preferred embodiments, the fragment or variant of an antibody that specifically binds a Therapeutic protein and that corresponds to a Therapeutic protein portion of an albumin fusion protein comprises, or alternatively consists of, an scFv comprising the VH domain of the Therapeutic antibody, linked to the VL domain of the therapeutic antibody by a peptide linker such as (Gly4Ser)3(SEQ ID NO:36).
Immunophenotyping
The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) ray be utilized for immunophenotyping of cell lines and biological samples. Therapeutic proteins of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies (or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies (or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Characterizing Antibodies that Bind a Therapeutic Protein and Albumin Fusion Proteins Comprising a Fragment or Variant of an Antibody that Binds a Therapeutic Protein
The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) may be characterized in a variety of ways. In particular, Albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for the ability to specifically bind to the same antigens specifically bound by the antibody that binds a Therapeutic protein corresponding to the antibody that binds a Therapeutic protein portion of the albumin fusion protein using techniques described herein or routinely modifying techniques known in the art.
Assays for the ability of the antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) to (specifically) bind a specific protein or epitope may be performed in solution (e.g., Houghten, Bio/Techniques 13:412-421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)) (each of these references is incorporated herein in its entirety by reference). The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) may also be assayed for their specificity and affinity for a specific protein or epitope using or routinely modifying techniques described herein or otherwise known in the art.
The albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be assayed for cross-reactivity with other antigens (e.g., molecules that have sequence/structure conservation with the molecule(s) specifically bound by the antibody that binds a Therapeutic protein (or fragment or variant thereof) corresponding to the Therapeutic protein portion of the albumin fusion protein of the invention) by any method known in the art.
Immunoassays which can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding an antibody of the invention or albumin fusion protein of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein (or fragment or variant thereof) to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C., adding protein A and/or protein G sepharose beads (or beads coated with an appropriate anti-iditoypic antibody or anti-albumin antibody in the case when an albumin fusion protein comprising at least a fragment or variant of a Therapeutic antibody) to the cell lysate, incubating for about an hour or more at 40 degrees C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody or albumin fusion protein of the invention to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody or albumin fusion protein to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), applying the antibody or albumin fusion protein of the invention (diluted in blocking buffer) to the membrane, washing the membrane in washing buffer, applying a secondary antibody (which recognizes the antibody or albumin fusion protein, e.g., an anti-human serum albumin antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the antibody or albumin fusion protein (comprising at least a fragment or variant of an antibody that binds a Therapeutic protein) of the invention conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound or non-specifically bound albumin fusion proteins, and detecting the presence of the antibody or albumin fusion proteins specifically bound to the antigen coating the well. In ELISAs the antibody or albumin fusion protein does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody or albumin fusion protein, respectively) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, antibody or the albumin fusion protein may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an albumin fusion protein to a protein, antigen, or epitope and the off-rate of an antibody- or albumin fusion protein-protein/antigen epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody or albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody or albumin fusion protein of the present invention for a specific protein, antigen, or epitope and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second protein that binds the same protein, antigen or epitope as the antibody or albumin fusion protein, can also be determined using radioimmunoassays. In this case, the protein, antigen or epitope is incubated with an antibody or albumin fusion protein of the present invention conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second protein that binds the same protein, antigen, or epuitope as the albumin fusion protein of the invention.
In a preferred embodiment, BIAcore kinetic analysis is used to determine the binding on and off rates of antibody or albumin fusion proteins of the invention to a protein, antigen or epitope. BIAcore kinetic analysis comprises analyzing the binding and dissociation of antibodies, albumin fusion proteins, or specific polypeptide, antigens or epitopes from chips with immobilized specific polypeptides, antigens or epitopes, antibodies or albumin fusion proteins, respectively, on their surface.
Therapeutic Uses
The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein to an animal, preferably a Mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein), nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein), albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein, and nucleic acids encoding such albumin fusion proteins. The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a Therapeutic protein, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a Therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein to an animal, preferably a mammal, and, most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions, and/or as described elsewhere herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a Therapeutic protein and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a Therapeutic protein, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a Therapeutic protein includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be provided in pharmaceutically acceptable compositions as known in the aft or as described herein.
A summary of the ways in which the antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be used therapeutically includes binding Therapeutic proteins locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein for diagnostic, monitoring or therapeutic purposes without undue experimentation.
The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokinesor hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention or albumin fusion proteins of the invention comprising at least a fragment or variant of an antibody that binds a Therapeutic protein may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against Therapeutic proteins, fragments or regions thereof, (or the albumin fusion protein correlate of such an antibody) for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include dissociation constants or Kd's less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M. More preferred binding affinities include those with a dissociation constant or Kd less than 5×10−5 M, 10−5 M, 5×10−6 M, 10−6 M, 5×10−7 M, 107 M, 5×10−8 M or 10−8 M. Even more preferred binding affinities include those with a dissociation constant or Kd less than 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10-12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M.
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding antibodies that bind Therapeutic proteins or albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect
Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described in more detail elsewhere in this application.
Demonstration of Therapeutic or Prophylactic Activity
The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Therapeutic Prophylactic Administration and Composition
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, arid oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,
Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents; or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tararic acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a Therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
Diagnosis and Imaging
Labeled antibodies and derivatives and analogs thereof that bind a Therapeutic protein (or fragment or variant thereof) (including albumin fusion proteins comprising at least a fragment or variant of an antibody that binds a Therapeutic protein), can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of Therapeutic protein. The invention provides for the detection of aberrant expression of a Therapeutic protein, comprising (a) in assaying the expression of the Therapeutic protein in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed Therapeutic protein expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the Therapeutic protein in cells or body fluid of an individual using one or more antibodies specific to the Therapeutic protein or albumin fusion proteins comprising at least a fragment of variant of an antibody specific to a Therapeutic protein, and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed Therapeutic protein gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Antibodies of the invention or albumin fusion proteins comprising at least a fragment of variant of an antibody specific to a Therapeutic protein can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One facet of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a Therapeutic protein in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the Therapeutic protein is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the Therapeutic protein. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody, antibody fragment, or albumin fusion protein comprising at least a fragment or variant of an antibody that binds a Therapeutic protein will then preferentially accumulate at the location of cells which contain the specific Therapeutic protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission to mography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Kits
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody, preferably a purified antibody, in-one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate sa radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment; binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support in a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagentis reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, Mo.).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Albumin Fusion Proteins
The present invention relates generally to albumin fusion proteins and methods of treating, preventing, or ameliorating diseases or disorders. As used herein, “albumin fusion protein” refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a Therapeutic protein (or fragment or variant thereof). An albumin fusion protein of the invention comprises at least a fragment or variant of a Therapeutic protein and at least a fragment or variant of human serum albumin, which are associated with one another, preferably by genetic fusion (i.e., the albumin fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a Therapeutic protein is joined in-frame with a polynucleotide encoding all or a portion of albumin) or chemical conjugation to one another. The Therapeutic protein and albumin protein, once part of the albumin fusion protein, may be referred to as a “portion”, “region” or “moiety” of the albumin fusion protein.
In one embodiment, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein (e.g., as described in Table 1) and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active fragment of a Therapeutic protein and a serum albumin protein. In other embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a biologically active and/or therapeutically active variant of a Therapeutic protein and a serum albumin protein. In preferred embodiments, the serum albumin protein component of the albumin fusion protein is the mature portion of serum albumin.
In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein, and a biologically active and/or therapeutically active fragment of serum albumin. In further embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, a Therapeutic protein and a biologically active and/or therapeutically active variant of serum albumin. In preferred embodiments, the Therapeutic protein portion of the albumin fusion protein is the mature portion of the Therapeutic protein.
In further embodiments, the invention provides an albumin fusion protein comprising or alternatively consisting of, a biologically active and/or therapeutically active fragment or variant of a Therapeutic protein and a biologically active and/or therapeutically active fragment or variant of serum albumin. In preferred embodiments, the invention provides an albumin fusion protein comprising, or alternatively consisting of, the mature portion of a Therapeutic protein and the mature portion of serum albumin.
Preferably, the albumin fusion protein comprises HA as the N-terminal portion, and a Therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion, and a Therapeutic prote in as the N-terminal portion may also be used.
In other embodiments, the albumin fusion protein has a Therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are the sanne Therapeutic proteins. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins. In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins which may be used to treat or prevent the same disease, disorder, or condition (e.g. as listed in the “Preferred Indication Y” column of Table 1). In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins which may be used to treat or prevent diseases or disorders (e.g. as listed in the “Preferred Indication Y” column of Table 1) which are known in the art to commonly occur in patients simultaneously.
In addition to albumin fusion protein in which the albumin portion is fused N-terminal and/or C-terminal of the Therapeutic protein portion, albumin fusion proteins of the invention may also be produced by inserting the Therapeutic protein or peptide of interest (e.g., a Therapeutic protein X as diclosed in Table 1, or an antibody that binds a Therapeutic protein or a fragment or variant thereof) into an internal region of HA. For instance, within the protein sequence of the HA molecule a number of loops or turns exist between the end and beginning of α-helices, which are stabilized by disulphide bonds (see FIGS. 9-11). The loops, as determined from the crystal structure of HA (FIG. 13) (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E to 1E7I and 1UOR) for the most part extend away from the body of the molecule. These loops are useful for the insertion, or internal fusion, of therapeutically active peptides, particularly those requiring a secondary structure to be functional, or Therapeutic proteins, to essentially generate an albumin molecule with specific biological activity.
Lops in human albumin structure into which peptides or polypeptides may be inserted to generate albumin fusion proteins of the invention include: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys50G-Thr566. In more preferred embodiments, peptides or polypeptides are inserted into the Val54-Asn61, Gin170-Ala176, and/or Lys560-Thr566 loops of mature human albumin (SEQ ID NO: 18).
Peptides to be inserted may be derived from either phage display or synthetic peptide libraries screened for specific biological activity or from the active portions of a molecule with the desired function. Additionally, random peptide libraries may be generated within particular loops or by insertions of randomized peptides into particular loops of the HA molecule and in which all possible combinations of amino acids are represented.
Such library(s) could be generated on HA or domain fragments of HA by one of the following methods:

    
    
        (a) randomized mutation of amino acids within one or more peptide loops of HA or HA domain fragments. Either one, more or all the residues within a loop could be mutated in this manner (for example see FIG. 10a);
        (b) replacement of, or insertion into one or more loops of HA or HA domain fragments (i.e., internal fusion) of a randomized peptide(s) of length Xl (where X is an amino acid and n is the number of residues (for example see FIG. 10b);
        (c) N-, C- or N- and C-terminal peptide/protein fusions in addition to (a) and/or (b).
    
    


The HA or HA domain fragment may also be made multifunctional by grating the peptides derived from different screens of different loops against different targets into the same HA or HA domain fragment.
In preferred embodiments, peptides inserted into a loop of human serum albumin are peptide fragments or peptide variants of the Therapeutic proteins disclosed in Table 1. More particularly, the invention encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids in length inserted into a loop of human serum albumin. The invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the N-terminus of human serum albumin. The invention also encompasses albumin fusion proteins which comprise peptide fragments or peptide variants at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids fused to the C-terminus of human serum albumin.
Generally, the albumin fusion proteins of the invention may have one HA-derived region and one Therapeutic protein-derived region. Multiple regions of each protein, however, may be used to make an albumin fusion protein of the invention. Similarly, more than one Therapeutic protein may be used to make an albumin fusion protein of the invention. For instance, a Therapeutic protein may be fused to both the N- and C-terminal ends of the HA. In such a configuration, the Therapeutic protein portions may be the same or different Therapeutic protein molecules. The structure of bifunctional albumin fusion proteins may be represented as: X-HA-Y or Y-HA-X.
For example, an anti-BLySTM scFv-HA-IFNα-2b fusion may be prepared to modulate the immune response to IFNα-2b by anti-BLySTM scFv. An alternative is making a bi (or even multi) functional dose of HA-fusions e.g. HA-IFNα-2b fusion mixed with HA-anti-BLySTM scFv fusion or other HA-fusions in various ratio's depending on function, half-life etc.
Bi- or multi-functional albumin fusion proteins may also be prepared to target the Therapeutic protein portion of a fusion to a target organ or cell type via protein or peptide at the opposite terminus of HA.
As an alternative to the fusion of known therapeutic molecules, the peptides could be obtained by screening libraries constructed as fusions to the N-, C- or N- and C-termini of HA, or domain fragment of HA, of typically 6, 8, 12, 20 or 25 or Xn (where X is an amino acid (aa) and n equals the number of residues) randomized amino acids, and in which all possible combinations of amino acids were represented. A particular advantage of this approach is that the peptides may be selected in situ on the HA molecule and the properties of the peptide would therefore be as selected for rather than, potentially, modified as might be the case for a peptide derived by any other method then being attached to HA.
Additionally, the albumin fusion proteins of the invention may include a linker peptide between the fused portions to provide greater physical separation between the moieties and thus maximize the accessibility of the Therapeutic protein portion, for instance, for binding to its cognate receptor. The linker peptide may consist of amino acids such that it is flexible or more rigid.
The linker sequence may be cleavable by a protease or chemically to yield the growth hormone related moiety. Preferably, the protease is one which is produced naturally by the host, for example the S. cerevisiae protease kex2 or equivalent proteases.
Therefore, as described above, the albumin fusion proteins of the invention may have the following formula R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is at least one Therapeutic protein, peptide or polypeptide sequence, and not necessarily the same Therapeutic protein, L is a linker and R2 is a serum albumin sequence.
In preferred embodiments, Albumin fusion proteins of the invention comprising a Therapeutic protein have extended shelf life compared to the shelf life the same Therapeutic protein when not fused to albumin. Shelf-life typically refers to the time period over which the therapeutic activity of a Therapeutic protein in solution or in some other storage formulation, is stable without undue loss of therapeutic activity. Many of the Therapeutic proteins are highly labile in their unfused state. As described below, the typical shelf-life of these Therapeutic proteins is markedly prolonged upon incorporation into the albumin fusion protein of the invention.
Albumin fusion proteins of the invention with “prolonged” or “extended” shelf-life exhibit greater therapeutic activity relative to a standard that has been subjected to the same storage and handling conditions. The standard may be the unfused full-length Therapeutic protein. When the Therapeutic protein portion of the albumin fusion protein is an analog, a variant, or is otherwise altered or does not include the complete sequence for that protein, the prolongation of therapeutic activity may alternatively be compared to the unfused equivalent of that analog, variant, altered peptide or incomplete sequence. As an example, an albumin fusion protein of the invention may retain greater than about 100% of the therapeutic activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the therapeutic activity of a standard when subjected to the same storage and handling conditions as the standard when compared at a given time point.
Shelf-life may also be assessed in terms of therapeutic activity remaining after storage, normalized to therapeutic activity when storage began. Albumin fusion proteins of the invention with prolonged or extended shelf-life as exhibited by prolonged or extended therapeutic activity may retain greater than about 50% of the therapeutic activity, about 60%, 70%, 80%, or 90% or more of the therapeutic activity of the equivalent unfused Therapeutic protein when subjected to the same conditions. For example, as discussed in Example 1, an albumin fusion protein of the invention comprising hGH fused to the full length HA sequence may retain about 80% or more of its original activity in solution for periods of up to 5 weeks or more under various temperature conditions.
Expression of Fusion Proteins
The albumin fusion proteins of the invention may be produced as recombinant molecules by secretion from yeast, a microorganism such as a bacterium, or a human or animal cell line. Preferably, the polypeptide is secreted from the host cells. We have found that, by fusing the hGH coding sequence to the HA coding sequence, either to the 5′ end or 3′ end, it is possible to secrete the albumin fusion protein from yeast without the requirement for a yeast-derived pro sequence. This was surprising, as other workers have found that a yeast derived pro sequence was needed for efficient secretion of hGH in yeast.
For example, Hiramatsu et al. (Appl Environ Microbiol 56:2125 (1990); Appl Environ Microbiol 57:2052 (1991)) found that the N-terminal portion of the pro sequence in the Mucor pusillus rennin pre-pro leader was important. Other authors, using the MFα-1 signal, have always included the MFα-1 pro sequence when secreting hGH. The pro sequences were believed to assist in the folding of the hGH-by acting as an intramolecular chaperone. The present invention shows that Ha or fragments of HA can perform a similar function.
Hence, a particular embodiment of the invention comprises a DNA construct encoding a signal sequence effective for directing secretion in yeast, particularly a yeast-derived signal sequence (especially one which is homologous to the yeast host), and the fused molecule of the first aspect of the invention, there being no yeast-derived pro sequence between the signal and the mature polypeptide.
The Saccharomyces cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.
Conjugates of the kind prepared by Poznansky et al., (FEBS Lett 239:18 (1988)), in which separately-prepared polypeptides are joined by chemical cross-linking, are not contemplated.
The present invention also includes a cell, preferably a yeast cell transformed to express an albumin fusion protein of the invention. In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged away. Many expression systems are known and may be used, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris, filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
Preferred yeast strains to be used in the production of albumin fusion proteins are D88, DXY1 and BXP10. D88 [leu2-3, leu2-122, can1, pra1, ubc4] is a derivative of paren strain AH22his+ (also known as DB1; see, e.g., Sleep et al. Biotechnology 8:42-46 (1990)). The strain contains a leu2 mutation which allows for auxotropic selection of 2 micron-based plasmids that contain the LEU2 gene. D88 also exhibits a derepression of PRB1 in glucose excess. The PRB1 promoter is normally controlled by two checkpoints that monitor glucose levels and growth stage. The promoter is activated in wild type yeast upon glucose depletion and entry into stationary phase. Strain D88 exhibits the repression by glucose but maintains the induction upon entry into stationary phase. The PRA1 gene encodes a yeast vacuolar protease, YscA endoprotease A, that is localized in the ER. The UBC4 gene is in the ubiquitination pathway and is involved in targeting short lived and abnormal proteins for ubiquitin dependant degradation. Isolation of this ubc4 mutation was found to increase the copy number of an expression plasmid in the cell and cause an increased level of expression of a desired protein expressed from the plasmid (see, e.g., International Publication No. WO99/00504, hereby incorporated in its entirety by reference herein).
DXY1, a derivative of D88, has the following genotype: [leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3]. In addition to the mutations isolated in D88, this strain also has a knockout of the YAP3 protease. This protease causes cleavage of mostly di-basic residues (RR, RK, KR, KK) but can also promote cleavage at single basic residues in proteins. Isolation of this yap3 mutation resulted in higher levels of full length HSA production (see, e.g., U.S. Pat. No. 5,965,386 and Kerry-Williams et al., Yeast 14:161-169 (1998), hereby incorporated in their entireties by reference herein).
BXP10 has the following genotype: leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3. In addition to the mutations isolated in DXY1, this strain also has a knockout of the PMT1 gene and the HSP150 gene. The PMT1 gene is a member of the evolutionarily conserved family of dolichyl-phosphate-D-mannose protein O-niannosyltransferases (Pmts). The transmembrane topology of Pmt1p suggests that it is an integral membrane protein of the endoplasmic reticulum with a role in O-linked glycosylation. This mutation serves to reduce/eliminate clinked glycosylation of HSA fusions (see, e.g., International Publication No. WO00/44772, hereby incorporated in its entirety by reference herein. Studies revealed that the Hsp150 protein is inefficiently separated from rHA by ion exchange chromatography. The mutation in the HSP150 gene removes a potential contaminant that has proven difficult to remove by standard purification techniques. See, e.g., U.S. Pat. No. 5,783,423, hereby incorporated in its entirety by reference herein.
The desired protein is produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid. The yeasts are transformed with a coding sequence for the desired protein in any of the usual ways, for example electroporation. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
Successfully transformed cells, i.e., cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.
Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
Preferred vectors for making albumin fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA which are described in detail in Example 2. FIG. 4 shows a map of the pPPC0005 plasmid that can be used as the base vector into which polynucleotides encoding Therapeutic proteins may be cloned to form HA-fusions. It contains a PRB1 S. cerevisiae promoter (PRB1p), a Fusion leader sequence (FL), DNA encoding HA (rHA) and an ADH1 S. cerevisiae terminator sequence. The sequence of the fusion leader sequence consists of the first 19 amino acids of the signal peptide of human serum albumin (SEQ ID NO:29) and the last five amino acids of the mating factor alpha 1 promoter (SLDKR, see EP-A-387 319 which is hereby incorporated by reference in its entirety.
The plasmids, pPPC0005, pScCHSA, pScNHSA, and pC4:HSA were deposited on Apr. 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 and given accession numbers ATCC, and, respectively. Another vector useful for expressing an albumin fusion protein in yeast the pSAC35 vector which is described in Sleep et al., BioTechnology 8:42 (1990) which is hereby incorporated by reference in its entirety.
A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alterative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, _-single-stranded termini with their 3′5′-exonucleolytic activities, and fill in recessed 3′-ends with their polymerizing activities.
The combination of these activities therefore generates bluntnded DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including Intentional Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA in accordance with the invention, if, for example, HA variants are to be prepared, is to use the polymerase chain reaction as disclosed by Saiki et al. (1988) Science 239, 487-491. In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
Exemplary genera of yeast contemplated to be useful in the practice of the present invention as hosts for expressing the albumin fusion proteins are Pichia (formerly classified as Hansenula), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromnyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. Preferred genera are those selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii. 
Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K. marxianus. A suitable Torulaspora species is T. delbrueckii. Examples of Pichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P. pastoris. Methods for the transformation of S. cerevisiae are taught generally in EP 251 744, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference.
Preferred exemplary species of Saccharomyces include S. cerevisiae, S. italicus, S. diastaticus, and Zygosaccharomyces rouxii. Preferred exemplary species of Kluyveromyces include K. fragilis and K. lactis. Preferred exemplary species of Hansenula include H. polymorpha (now Pichia angusta), H. anomala (now Pichia anomala), and Pichia capsulata. Additional preferred exemplary species of Pichia include P. pastoris. Preferred exemplary species of Aspergillus include A. niger and A. nidulans. Preferred exemplary species of Yarrowia include Y. lipotytica. Many preferred yeast species are available from the ATCC. For example, the following preferred yeast species are available from the ATCC and are useful in the expression of albumin fusion proteins: Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3 mutant (ATCC Accession No. 4022731); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 hsp150 mutant (ATCC Accession No. 4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 pmtl mutant (ATCC Accession No. 4023792); Saccharomyces cerevisiae Hansen, teleomorph (ATCC Accession Nos. 20626; 44773; 44774; and 62995); Saccharomyces diastanicus Andrews et Gilliland ex van der Walt, teleomorph (ATCC Accession No. 62987); Kluyveromyces lactis (Dombrowski) van der Walt, teleomorph (ATCC Accession No. 76492); Pichia angusta (Teunisson et al.) Kurtzman, teleomorph deposited as Hansenula polymorpha de Morais et Maia, teleomorph (ATCC Accession No. 26012); Aspergillus niger van Tieghem, anamorph (ATCC Accession No. 9029); Aspergillus niger van Tieghem, anamorph (ATCC Accession No. 16404); Aspergillus nidulans (Eidam) Winter, anamorph (ATCC Accession No. 48756); and Yarrowia lipolytica (Wickerham et al.) van der Walt et von Arx, teleomorph (ATCC Accession No. 201847).
Suitable promoters for S. cerevisiae include those associated with the PGKI gene, GAL1 or GAL10 genes, CYCI, PHO5, TRPI, ADHI, ADH2, the genes for lyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphiofructokinase, triose phosphate isomerase, phosphoglucose isbmerase, glucokinase, alpha-mating factor pheromone, [a mating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI promoter, and hybrid promoters involving hybrids of parts of 5′ regulatory regions with parts of 5′ regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).
Convenient regulatable promoters for use in Schizosaccharomyces pombe are the thiamine-repressible promoter from the nmt gene as described by Maundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucose repressible jbpl gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.
Methods of transforming Pichia for expression of foreign genes are taught in, for example, Cregg et al. (1993), and various Phillips patents (e.g. U.S. Pat. No. 4,857,467, incorporated herein by reference), and Pichia expression kits are commercially available from Invitrogen BV, Leek, Netherlands, and Invitrogen Corp., San Diego, Calif. Suitable promoters include AOXI and AOX2. Gleeson et al. (1986) J. Gen. Microbiol. 132, 3459-3465 include information on Hiansenuta vectors and transformation, suitable promoters being MOX1 and FMD1; whilst EP 361 991, Fleer et al. (1991) and other publications from Rhone-Poulenc Rorer teach how to express foreign proteins in Kluyveromyces spp., a suitable promoter being PGKI.
The transcription termination signal is preferably the 3′ flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation. Suitable 3′ flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADHI gene is preferred.
The desired albumin fusion protein may be initially expressed with a secretion leader sequence, which may be any leader effective in the yeast chosen. Leaders useful in S. cerevisiae include that from the mating factor alpha polypeptide (MFα-1) and the hybrid leaders of EP-A-387 319. Such leaders (or signals) are cleaved by the yeast before the mature albumin is released into the surrounding medium. Further such leaders include those of S. cerevisiae invertase (SUC2) disclosed in JP 62-096086 (granted as 911036516), acid phosphatase (PH05), the pre-sequence of MFα-1, 0 glucanase (BGL2) and killer toxin; S. asicus glucoamylase II; S. carlsbergensis α-galactosidase (MEL1); K. lactis killer toxin; and Candida glucoamylase. 
Additional Methods of Recombinant and Synthetic Production of Albumin Fusion Proteins
The present invention also relates to vectors containing a polynucleotide encoding an albumin fusion protein of the present invention, host cells, and the production of albumin fusion proteins by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides encoding albumin fusion proteins of the invention may be joined to a vector containing a selectable marker for propagation in a host Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutarine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimunum cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastons (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukayotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.)., Other suitable vectors will be readily apparent to the skilled artisan.
In one embodiment, polynucleotides encoding an albumin fusion protein of the invention may be fused to signal sequences which will direct the localization of a protein of the invention to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a protein of the invention from a prokaryotic or eukaryotic cell. For example, in E. coli, one may wish to direct the expression of the protein to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the albumin fusion proteins of the invention may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit, and the signal sequence of alkaline phosphatase. Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs. In a specific embodiment, polynucleotides albumin fusion proteins of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
Examples of signal peptides that may be fused to an albumin fusion protein of the invention in order to direct its secretion in mammalian cells include, but are not limited to, the MPIF-1 signal sequence (e.g., amino acids 1-21 of GenBank Accession number AAB51134), the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:34), and a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:35). A suitable signal sequence that may be used in conjunction with baculoviral expression systems is the gp67 signal sequence (e.g., amino acids 1-19 of GenBank Accession Number AAA72759).
Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukayotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
In addition to encompassing host cells containing the vector, constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence corresponding to a Therapeutic protein may be replaced with an albumin fusion protein corresponding to the Therapeutic protein), and/or to include genetic material (e.g., heterologous polynucleotide sequences such as for example, an albumin fusion protein of the invention corresponding to the Therapeutic protein may be included). The genetic material operably associated with the endogenous polynucleotide may activate, alter, and/or amplify endogenous polynucleotides.
In addition, techniques known in the art may be used to operably associate heterologous polynucleotides (e.g., polynucleotides encoding an albumin protein, or a fragment or variant thereof) and/or heterologous control regions (e.g., promoter and/or enhancer) with endogenous polynucleotide sequences encoding a Therapeutic protein via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication Number. WO 96/29411; International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijistra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
Albumin fusion proteins of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
In preferred embodiments the albumin fusion proteins of the invention are purified using Anion Exchange Chromatography including, but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE columns.
In specific embodiments the albumin fusion proteins of the invention are purified using Cation Exchange Chromatography including, but not limited to, SP-sepharose, CM sepharbse, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM columns and their equivalents and comparables.
In specific embodiments the albumin fusion proteins of the invention are purified using Hydrophobic Interaction Chromatography including, but not limited to, Phenyl, Butyl, Methyl, Octyl, Hexyl-sepharose, poros Phenyl, Butyl, Methyl, Octyl, Hexyl, Toyopearl Phenyl, Butyl, Methyl, Octyl, Hexyl Resource/Source Phenyl, Butyl, Methyl, Octyl, Hexyl, Fractogel Phenyl, Butyl, Methyl, Octyl, Hexyl columns and their equivalents and comparables.
In specific embodiments the albumin fusion proteins of the invention are purified using Size Exclusion Chromatography including, but not limited to, sepharose S100, S200, S300, superdex resin columns and their equivalents and comparables.
In specific embodiments the albumin fusion proteins of the invention are purified using Affinity Chromatography including, but not limited to, Mimetic Dye affinity, peptide affinity and antibody affinity columns that are selective for either the HSA or the “fusion target” molecules.
In preferred embodiments albumin fusion proteins of the invention are purified using one or more Chromatography, methods listed above. In other preferred embodiments, albumin fusion proteins of the invention are purified using one or more of the following Chromatography columns, Q sepharose FF column, SP Sepharose FF column, Q Sepharose High Performance Column, Blue Sepharose FF column, Blue Column, Phenyl Sepharose FF column, DEAE Sepharose FF, or Methyl Column.
Additionally, albumin fusion proteins of the invention may be purified using the process described in PCT International Publication WO 00/44772 which is herein incorporated by reference in its entirety. One of skill in the art could easily modify the process described therein for use in the purification of albumin fusion proteins of the invention.
Albumin fusion proteins of the present invention may be recovered from: products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, albumin fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastors is used to express albumin fusion proteins of the invention in a eukaryotic system. Pichia pastons is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding an albumin fusion protein of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, PPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide encoding an albumin fusion protein of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition, albumin fusion proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids-include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, balanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses albumin fusion proteins of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational miodifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The albumin fusion proteins may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (121I, 123I, 125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In, 112In, 113mIn, 115mIn), technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, and 97Ru.
In specific embodiments, albumin fusion proteins of the present invention or fragments or variants thereof are attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177Lu, 90Y, 166Ho, and 153Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is 90Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
As mentioned, the albumin fusion proteins of the invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phdsphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemizaton, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Albumin fusion proteins of the invention and antibodies that bind a Therapeutic protein or fragments or variants thereof can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
Further, an albumin fusion protein of the invention may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromfomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, B-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptoic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Techniques for conjugating such therapeutic moiety to proteins (e.g., albumin fusion proteins) are well known in the art.
Albumin fusion proteins may also be attached to solid supports, which are particularly useful for immunoassays or purification of polypeptides that are bound by, that bind to, or associate with albumin fusion proteins of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Albumin fusion proteins, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
In embodiments where the albumin fusion protein of the invention comprises only the VH domain of an antibody that binds a Therapeutic protein, it may be necessary and/or desirable to coexpress the fusion protein with the VL domain of the same antibody that binds a Therapeutic protein, such that the VH-albumin fusion protein and VL protein will associate (either covalently or non-covalently) post-translationally.
In embodiments where the albumin fusion protein of the invention comprises only the VL domain of an antibody that binds a Therapeutic protein, it may be necessary and/or desirable to coexpress the fusion protein with the VH domain of the same antibody that binds a Therapeutic protein, such that the VL-albumin fusion protein and VH protein will associate (either covalently or non-covalently) post-translationally.
Some Therapeutic antibodies are bispecific antibodies, meaning the antibody that binds a Therapeutic protein is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. In order to create an albumin fusion protein corresponding to that Therapeutic protein, it is possible to create an albumin fusion protein which has an scFv fragment fused to both the N- and C-terminus of the albumin protein moiety. More particularly, the scFv fused to the N-terminus of albumin would correspond to one of the heavy/light (VH/VL) pairs of the original antibody that binds a Therapeutic protein and the scFv fused to the C-terminus of albumin would correspond to the other heavy/light (VH/VL) pair of the original antibody that binds a Therapeutic protein.
Also provided by the invention are chemically modified derivatives of the albumin fusion proteins of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The albumin fusion proteins may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a Therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000; 17,500, 18,000, 18,500, 19,000, 19,500; 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutaric acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the albumin fusion proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the albumin fusion protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by, reference.
One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-triflubreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each albumin fusion protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
The presence and quantity of albumin fusion proteins of the invention may be determined using ELISA, a well known immunoassay known in the art. In one ELISA protocol that would be useful for detecting/quantifying albumin fusion proteins of the invention, comprises the steps of coating an ELISA plate with an anti-human serum albumin antibody, blocking the plate to prevent non-specific binding, washing the ELISA plate, adding a solution containing the albumin fusion protein of the invention (at one or more different concentrations), adding a secondary anti-Therapeutic protein specific antibody coupled to a detectable label (as described herein or otherwise known in the art), and detecting the presence of the secondary antibody. In an alternate version of this protocol, the ELISA plate might be coated with the anti-Therapeutic protein specific antibody and the labeled secondary reagent might be the anti-human albumin specific antibody.
Uses of the Polynucleotides
Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful to produce the albumin fusion proteins of the invention. As described in more detail below, polynucleotides of the invention (encoding albumin fusion proteins) may be used in recombinant DNA methods useful in genetic engineering to make cells, cell lines, or tissues that express the albumin fusion protein encoded by the polynucleotides encoding albumin fusion proteins of the invention.
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled “Gene Therapy”, and Examples 17 and 18).
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
Albumin fusion proteins of the invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry assays).
Albumin fusion proteins can be used to assay levels of polypeptides in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Albumin fusion proteins of the invention can also be detected in vivo by imaging.
Labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR) or electron spin relaxtion (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients given to a cell line expressing the albumin fusion protein of the invention.
An albumin fusion protein which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc, (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F, 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr; 105Rh, 97Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at locations in the body (e.g., organs, cells, extracellular spaces or matrices) where one or more receptors, ligands or substrates (corresponding to that of the Therapeutic protein used to make the albumin fusion protein of the invention) are located. Alternatively, in the case where the albumin fusion protein comprises at least a fragment or variant of a Therapeutic antibody, the labeled albumin fusion protein will then preferentially accumulate at the locations in the body (e.g., organs, cells, extracellular spaces or matrices) where the polypeptides/epitopes corresponding to those bound by the Therapeutic antibody (used to make the albumin fusion protein of the invention) are located. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)). The protocols described therein could easily be modified by one of skill in the art for use with the albumin fusion proteins of the invention.
In one embodiment, the invention provides a method for the specific delivery of albumin fusion proteins of the invention to cells by administering albumin fusion proteins of the invention (e.g., polypeptides encoded by polynucleotides encoding albumin fusion proteins of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a Therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering albumin fusion proteins of the invention in association with toxins or cytotoxic prodrugs.
By “toxin” is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or other radioisotopes such as, for example, 103Pd, 133Xe, 131I, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, 90Yttrium, 117Tin, 186Rhenium, 166Holmium, and 188Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 90Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 111In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131I.
Techniques known in the art may be applied to label polypeptides of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
The albumin fusion proteins of the present invention art useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described herein under the section heading “Biological Activities,” below.
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a certain polypeptide in cells or body fluid of an individual using an albumin fusion protein of the invention; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Moreover, albumin fusion proteins of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
In particular, albumin fusion proteins comprising of at least a fragment or variant of a Therapeutic antibody can also be used to treat disease (as described supra and elsewhere herein). For example, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can bind, and/or neutralize the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds, and/or reduce overproduction of the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds. Similarly, administration of an albumin fusion protein comprising of at least a fragment or variant of a Therapeutic antibody can activate the polypeptide to which the Therapeutic antibody used to make the albumin fusion protein immunospecifically binds, by binding to the polypeptide bound to a membrane (receptor).
At the very least, the albumin fusion proteins of the invention of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Albumin fusion proteins of the invention can also be used to raise antibodies, which in turn may be used to measure protein expression of the Therapeutic protein, albumin protein, and/or the albumin fusion protein of the invention from a recombinant cell, as a way of assessing transformation of the host cell, or in a biological sample. Moreover, the albumin fusion proteins of the present invention can be used to test the biological activities described herein.
Diagnostic Assays
The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not-limited to, those described for each Therapeutic protein in the corresponding row of Table 1 and herein under the section headings “Immune Activity,” “Blood Related Disorders,” “Hyperproliferative Disorders,” “Renal Disorders,” “Cardiovascular Disorders,” “Respiratory Disorders,” “Anti-Angiogenesis Activity,” “Diseases at the Cellular Level,” “Wound Healing and Epithelial Cell Proliferation,” “Neural Activity and Neurological Diseases,” “Endocrine Disorders,” “Reproductive System Disorders,” “Infectious Disease,” “Regeneration,” and/or “Gastrointestinal Disorders,” infra.
For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding a polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome
By “assaying the expression level of the gene encoding a polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of a particular polypeptide (e.g. a polypeptide corresponding to a Therapeutic protein disclosed in Table 1) or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting abnormal expression of polypeptides that bind to, are bound by, or associate with albumin fusion proteins compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide that bind to are bound by, or associate with albumin fusion proteins of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying, polypeptide levels in a biological sample can occur using any art-known method.
Assaying polypeptide levels in a biological sample can occur using a variety of techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods. (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of interest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
For example, albumin fusion proteins may be used to quantitatively or qualitatively detect the presence of polypeptides that bind to, are bound by, or associate with albumin fusion proteins of the present invention. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled albumin fusion protein coupled with light microscopic, flow cytometric, or fluorimetric detection.
In a preferred embodiment, albumin fusion proteins comprising at least a fragment or variant of an antibody that immunospecifically binds at least a Therapeutic protein disclosed herein (e.g., the Therapeutic proteins disclosed in Table 1) or otherwise known in the art may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
The albumin fusion proteins of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of polypeptides that bind to, are bound by, or associate with an albumin fusion protein of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The albumin fusion proteins are preferably applied by overlaying the labeled albumin fusion proteins onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the polypeptides that bind to, are bound by, or associate with albumin fusion proteins, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Immunoassays and non-immunoassays that detect polypeptides that bind to, are bound by, or associate with albumin fusion proteins will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled albumin fusion protein of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
By “solid phase support or carrier” is intended any support capable of binding a polypeptide (e.g., an albumin fusion protein, or polypeptide that binds, is bound by, or associates with an albumin fusion protein of the invention.) Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polypeptide. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of albumin fusion protein may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
In addition to assaying polypeptide levels in a biological sample obtained from an individual, polypeptide can also be detected in vivo by imaging. For example, in one embodiment of the invention, albumin fusion proteins of the invention are used to image diseased or neoplastic cells.
Labels or markers for in vivo imaging of albumin fusion proteins of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the albumin fusion protein by labeling of nutrients of a cell line (or bacterial or yeast strain) engineered.
Additionally, albumin fusion proteins of the invention whose presence can be detected, can be administered. For example, albumin fusion proteins of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
A polypeptide-specific antibody or antibody fragment which has been labeled with an apropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 121In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled albumin fusion protein will then preferentially accumulate at the locations in the body which contain a polypeptide or other substance that binds to, is bound by or associates with an albumin fusion protein of the present invention. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
One of the ways in which an albumin fusion protein of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly. Publication, Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Albumin fusion proteins may also be radiolabelled and used in any of a variety of other immunoassays. For example, by radioactively labeling the albumin fusion proteins, it is possible to the use the albumin fusion proteins in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques. The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
It is also possible to label the albumin fusion proteins with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
The albumin fusion protein can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The albumin fusion proteins can also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged albumin fusion protein is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label albumin fusion proteins of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
Transgenic Organisms
Transgenic organisms that express the albumin fusion proteins of the invention are also included in the invention. Transgenic organisms are genetically modified organisms into which recombinant, exogenous or cloned genetic material has been transferred. Such genetic material is often referred to as a transgene. The nucleic acid sequence of the transgene may include one or more transcriptional regulatory sequences and other nucleic acid sequences such as introns, that may be necessary for optimal expression and secretion of the encoded protein. The transgene may be designed to direct the expression of the encoded protein in a manner that facilitates its recovery from the organism or from a product produced by the organism, e.g. from the milk, blood, urine, eggs, hair or seeds of the organism. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal. The transgene may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
The term “germ cell line transgenic organism” refers to a transgenic organism in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic organism to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic organisms. The alteration or genetic information may be foreign to the species of organism to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
A transgenic organism may be a transgenic animal or a transgenic plant. Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Pat. No. 5,489,743 and U.S. Pat. No. 5,602,307.
A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Pat. No. 4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat. No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Pat. No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4):1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) The Lancet 349(9049):405).
While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617; Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; Schnieke et al. (1997) Science 278(5346):2130-2133; and Amoah (1997) J. Animal Science 75(2):578-585).
To direct the secretion of the transgene-encoded protein of the invention into the milk of transgenic mammals, it may be put under the control of a promoter that is preferentially activated in mammary epithelial cells. Promoters that control the genes encoding milk proteins are preferred, for example the promoter for casein, beta lactoglobulin, whey acid protein, or lactalbumin (see, e.g., DiTullio (1992) BioTechnology 10:74-77; Clark et al. (1989) BioTechnology 7:487-492; Gorton et al. (1987) BioTechnology 5:1183-1187; and Soulier et al. (1992) FEBS Letts. 297:13). The transgenic mammals of choice would produce large volumes of milk and have long lactating periods, for example goats, cows, camels or sheep.
An albumin fusion protein of the invention can also be expressed in a transgenic plant, e.g. a plant in which the DNA transgene is inserted into the nuclear or plastidic genome. Plant transformation procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art (e.g., see Example 19). See, in general, Methods in Enzymology Vol. 153 (“Recombinant DNA Part D”). 1987, Wu and Grossman Eds., Academic Press and European Patent Application EP 693554. Methods for generation of genetically engineered plants are further described in U.S. Pat. No. 5,283,184, U.S. Pat. No. 5,482,852, and European Patent Application EP 693 554, all of which are hereby incorporated by reference.
Pharmaceutical or Therapeutic Compositions
The albumin fusion proteins of the invention or formulations thereof may be administered by any conventional method including parenteral (e.g. subcutaneous or intramuscular) injection or intravenous infusion. The treatment may consist of a single dose or a plurality of doses over a period of time.
While it is possible for an albumin fusion protein of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be “acceptable” in the sense of being compatible with the albumin fusion protein and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free. Albumin fusion proteins of the invention are particularly well suited to formulation in aqueous carriers such as sterile pyrogen free water, saline or other isotonic solutions because of their extended shelf-life in solution. For instance, pharmaceutical compositions of the invention may be formulated well in advance in aqueous form, for instance, weeks or months or longer time periods before being dispensed.
For example, wherein the Therapeutic protein is hGH, EPO, alpha-IFN or beta-IFN, formulations containing the albumin fusion protein may be prepared taking into account the extended shelf-life of the albumin fusion protein in aqueous formulations. As exhibited in Table 2, most Therapeutic proteins are unstable with short shelf-lives after formulation with an aqueous carrier. As discussed above, the shelf-life of many of these Therapeutic proteins are markedly increased or prolonged after fusion to HA.











TABLE 2






Tradename,


Storage Conditions of


Protein
Manufacturer
Route
Formulation
Non-Fusion Protein







Interferon,
Roferon-A,
sc
sol_n
4-8° C.


alpha-2a
Hoffmann-LaRoche
im
(vial or pre-filled





syringe)


Interferon,
Intron-A,
iv sc im
sol_n;
4-8° C.


alpha-2b
Schering Plough

powder + dil.
(all preps, before and






after dilution)


COMBO
Rebetron (Intron-A +
po
Rebetol capsule +


Interferon alpha-
Rebetol)
+
Intron-A injection


2b +
Schering Plough
sc


Ribavirin


Interferon,
Infergen
sc
sol_n
4-8° C.


Alphacon-1
Amgen


Interferon,
Wellferon,
sc
sol_n
4-8° C.


alpha-n1,
Wellcome
im
(with albumin _as


Lympho-


stabiizer_)


blastoid


Interferon,
Avonex,
im
powder + dil.
4-8° C.


beta-1a
Biogen

(with albumin)
(before and after dilution)






(Use within 3-6 h of






reconstitution)



Rebif,
sc
sol_n,



Ares-Serono

in pre-filled syringe



(Europe only)


Interferon,
Betaseron,
sc
powder + dil.
4-8° C.


beta-1b
Chiron

(with albumin)
(before and after dilution)



(Europe: Betaferon)


(Use within 3 h of






reconstitution)






Single use vials.


Interferon,
Actimmune,
sc

4-8° C.


Gamma-1b
InterMune


(before and after dilution)



Pharmaceuticals


(Use within 3 h of






reconstitution).


Growth
Genotropin,

powder/dil cartridges
4-8° C.


Hormone
Pharmacia Upjohn

(single or multi-use);
(before and after dilution);


(somatropin)


single use MiniQuick
single use MiniQuick





injector
Delivery Device should






be refrigerated until use.



Humatrope,
sc
powder + dil.
4-8° C.



Eli Lilly
im
(Vial or pen cartridge)
(before and after dilution)






(Use vials within 25 h,






cartridges within 28 d, of






reconstitution).



Norditropin,



Novo Nordisk



Pharmaceuticals



Nutropin,
sc
powder + dil.
4-8° C.



Genentech


(stable for 14 d after dil_n)






(all preps, before and after






dilution)



Nutropin AQ,
sc
sol_n
4-8° C.



Genentech


(Stable for 28 d after






1st use)



Nutropin Depot,
sc
microsphere suspension
4-8° C.



Genentech

as
Single use pkges. Dose





powder + dil.
1-2×/month (ProLease






micro-encapsulation






technol.)



Saizen,
sc
powder + dil.
Powder _should be stored



(Serono)
im

at RmTemp_. After






reconstitution store 4-






8° C. for up to 14 d.



Serostim,


Powder _should be stored



Serono


at Rm Temp_. After






reconstitution store in 4-






8° C. for up to 14 d.


hGH, with
Protropin,
sc
powder + dil.
4-8° C.


N-term. Met
Genentech
im

(all preps, before and


(somatrem)



after dilution)


Erythropoietin
Epogen,
iv
sol_n
4-8° C.


(Epoetin alfa)
Amgen
sc

(use within 21 d of first






use)






(Single & multi-dose






vials)



Procrit,
iv
sol_n
4-8° C.



Amgen
sc

(use within 21 d of






first use)






(Single & multi-dose






vials)









In instances where aerosol administration is appropriate, the albumin fusion proteins of the invention can be formulated as aerosols using standard procedures. The term “aerosol” includes any gas-borne suspended phase of an albumin fusion protein of the instant invention which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets of an albumin fusion protein of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer Aerosol also includes a dry powder composition of a compound of the instant invention suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delvery to the Respiratory Trac, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al,. (1992) Pharmacol. Toxcol. Methods 27:143-159.
The formulations of the invention are also typically non-immunogenic, in part, because of the use of the components of the albumin fusion protein being derived from the proper species. For instance, for human use, both the Therapeutic protein and albumin portions of the albumin fusion protein will typically be human. In some cases, wherein either component is non human-derived, that component may be humanized by substitution of key amino acids so that specific epitopes appear to the human immune system to be human in nature rather than foreign.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the albumin fusion protein with the carrier that constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation appropriate for the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules, vials or syringes and may be stored in a freezedried (lyophilised) condition requirins only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders. Dosage formulations may contain the Therapeutic protein portion at a lower molar concentration or lower dosage compared to the non-fused standard formulation for the Therapeutic protein given the extended serum half-life exhibited by many of the albumin fusion proteins of the invention.
As an example, when an albumin fusion protein of the invention comprises growth hormone as one or more of the Therapeutic protein regions, the dosage form can be calculated on the basis of the potency of the albumin fusion protein relative to the potency of hGH, while taking into account the prolonged serum half-life and shelf-life of the albumin fusion proteins compared to that of native hGH. Growth hormone is typically administered at 0.3 to 30.0 IU/kg/week, for example 0.9 to 12.0 IU/kg/week, given in three or seven divided doses for a year or more. In an albumin fusion protein consisting of full length HA fused to full length GH, an equivalent dose in terms of units would represent a greater weight of agent but the dosage frequency can be reduced, for example to twice a week, once a week or less.
Formulations or compositions of the invention may be packaged together with, or included in a kit with, instructions or a package insert referring to the extended shelf-life of the albumin fusion protein component. For instance, such instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the extended or prolonged shelf-life of the albumin fusion proteins of the invention. Such instructions or package inserts may also address the particular advantages of the albumin fusion proteins of the inventions, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions. As described above, formulations of the invention may be in aqueous form and may be stored under less than ideal circumstances without significant loss of therapeutic activity.
Albumin fusion proteins of the invention can also be included in nutraceuticals. For instance, certain albumin fusion proteins of the invention may be administered in natural products, including milk or milk product obtained from a transgenic mammal which expresses albumin fusion protein. Such compositions can also include plant or plant products obtained from a transgenic plant which expresses the albumin fusion protein. The albumin fusion protein can also be provided in powder or tablet form, with or without other known additives, carriers, fillers and diluents. Nutraceuticals are described in Scott Hegenhart, Food Product Design, December 1993.
The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of an albumin fusion protein of the invention or a polynucleotide encoding an albumin fusion protein of the invention (“albumin fusion polynucleotide”) in a pharmaceutically acceptable carrier.
The albumin fusion protein and/or polynucleotide will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the albumin fusion protein and/or polynucleotide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of the albumin fusion protein administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the albumin fusion protein is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
Albumin fusion proteins and/or polynucleotides can be are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal subcutaneous and intraarticular injection and infusion.
Albumin fusion proteins and/or polynucleotides of the invention are also suitably administered by sustained-release systems. Examples of sustained-release albumin fusion proteins and/or polynucleotides are administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray, “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Additional examples of sustained-release albumin fusion proteins and/or polynucleotides include suitable polymeric materials. (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).
Sustained-release albumin fusion proteins and/or polynucleotides also include liposomally entrapped albumin fusion proteins and/or polynucleotides of the invention (see generally. Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 317-327 and 353-365 (1989)). Liposomes containing the albumin fusion protein and/or polynucleotide are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
In yet an additional embodiment, the albumin fusion proteins and/or polynucle tides of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574(1989)).
Other controlled release systems are discussed in the review by Lancer (Science 249:1527-1533 (1990)).
For parenteral administration, in one embodiment, the albumin fusion protein and/or polynucleotide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
Generally, the formulations are prepared by contacting the albumin fusion protein and/or polynucleotide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
The albumin fusion protein is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Albumin fusion proteins and/or polynucleotides generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Albumin fusion proteins and/or polynucleotides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous albumin fusion protein and/or polynucleotide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized albumin fusion protein and/or polynucleotide using bacteriostatic Water-for-Injection.
In a specific and preferred embodiment, the Albumin fusion protein formulations comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. In another specific and preferred embodiment, the Albumin fusion protein formulations consists 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromole sodium octanoate/milligram of fusion protein, 15 micrograms/milliliter polysorbate 80, pH 7.2. The pH and buffer are chosen to match physiological conditions and the salt is added as a tonicifier. Sodium octanoate has been chosen due to its reported ability to increase the thermal stability of the protein in solution. Finally, polysorbate has been added as a generic surfactant, which lowers the surface tension of the solution and lowers non-specific adsorption of the albumin fusion protein to the container closure system.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the albumin fusion proteins and/or polynucleotides of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the albumin fusion proteins and/or polynucleotides may be employed in conjunction with other therapeutic compounds.
The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable preparations of Corynebacterium parvum. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with alum. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, Haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
The albumin fusion proteins and/or polynucleotides of the invention may be administered alone or in combination with other therapeutic agents. Albumin fusion protein and/or polynucleotide agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further-includes the separate administration of one of the compounds or agents given first, followed by the second.
In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADONTM), acenocoumarol (e.g., nicoumalone, SINTHROMETM), indan-1,3-dione, phenprocoumon (e.g., MARCUMARTM), ethyl biscoumace/tate (e.g., TROMEXANTM), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.
In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with thrombolytic drugs. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KABIKINASETM), antiresplace (e.g., EMINASETM), tissue plasminogen activator (t-PA, altevase; ACTIVASETM), urokinase (e.g., ABBOKINASETM) sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICARTM). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.
In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an in platelet drugs. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINETM), and ticlopidine (e.g., TICLIDTM).
In specific embodiments, the use of anti-coagulants, thrombolytic and/or and platelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention, diagnosis, and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with albumin fusion proteins and/or polynucleotides of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reduicing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodiaiysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
In certain embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddI), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine). NNRTIs that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, VIRAMUNETM (mevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz). Protease inhibitors that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir), INVIRASETM (saquinavir), and VIRACEPTTM (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with albumin fusion proteins and/or polynucleotides of the invention to treat AIDS and/or to prevent or treat HIV infection.
Additional NRTIs include LODENOSINETM (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACILTM (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIRTM (bis-POC PMPA, a PMPA prodrug Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGENTM (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′azido-2′,3′dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (see, International Publication No. WO 98/17281).
Additional NNRTIs include COACTINONTM (Emivirine/MKC442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINETM (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (see, International Publication No. WO 99/49836).
Additional protease inhibitors include LOPINAVIRTM (AEB378/r, Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIRTM (PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASETM (amprenavir; Glaxo Wellcome Inc.).
Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to g41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor: Trimeris).
Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibit fusion.
Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIRTM (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.
Additional antiretroviral agents include hydroxyurea-like compunds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOXTM (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).
Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear tanslocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.
Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKINTM (aldesleukin/L2-7001; Chiron), IL4, IL10, IL-12, and IL-13; interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as RemuneTM (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rap120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 93/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yana er al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268D and 50.1 anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (see, International Publication No. WO 98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester. (γ-GCE; WO 99/56764).
In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
In other embodiments, albumin fusion proteins and/or polynucleotides of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINE, ATOVAQUONETM, ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM, KETOCONAZOLETM, ACYCLOVIRTM, FAMCICOLVIRTM, PYRIMETHAMINETM, LEUCOVORINTM, NEUPOGENTM (filgrastim/G-CSF), and LEUKINETM (sargramostim/GM-CSF). In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, and/or ATOVAQUONETM to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or ETHAMBUTOLTM to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with RIFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with FLUCONAZOLETM, ITRACONAZOLETM, and/or KETOCONAZOLETM to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with PYRIMETHAMINETM and/or LEUCOVORINTM to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat or prevent an opportunistic bacterial infection.
In a further embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.
In other embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with immunestimulants. Immunostimulants that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, levamisole (e.g., ERGAMISOLTM), isoprinosine (e.g. INOSIPLEXTM), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).
In other embodiments, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with imnunosuppressive agents. Immunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells Other immunosuppressive agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININTM), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNETM, NEORALTM, SANGDYATM (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenotate motefil, of which the active metabolite is mycophenolic acid), IMURANTM (azathioprine), glucocoricosteroids, adrenocortical steroids such as DELTASONETM (prednisone) and HYDELTRASOLTM (prednisolone), FOLEXTM and MEXATETM (methotrxate), OXSORALEN-ULTRATM (methoxsalen) and RAPAMUNETM (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
In an additional embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, ATGAMTM (antithymocyte glubulin), and GAMIMUNETM. In a specific embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone), nonsteroidal anti-inflammatory drugs. (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life. Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tunasten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tunsgtate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxaolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem J. 236:475-480, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiornalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Loberzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.
Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)), an integrin alpha v beta 3 antagonist (C. Storard et al., J Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXIGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstrZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panrerin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.
Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositions of the invention include, but are not lmited to, AG-3340 (Agouron. La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aetema, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositions of the invention include, but are not Imited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositions of the invention include, but are not Imited to, Angiozyme (Riboziyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositions of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nudey, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).
In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.
In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encodin, an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, plateletderived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine arid Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil, 5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).
In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as RemicadeTM Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as AravaTM from Hoechst Marion Roussel), KineretTM (an IL-1 Receptor antagonist also known as anakinra from Amgen, Inc.)
In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
In another specific embodiment, the compositions of the invention are administered in combination ZevalinTM. In a further embodiment, compositions of the invention are administered with ZevalinTM and CHOP, or ZevalinTM and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. ZevalinTM may be associated with one or more radisotopes. Particularly preferred isotopes are 90Y and 111In.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with cytokines. Cytokines that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, albumin fusion proteins and/or polynucleotides of the invention may be administered with any interleukin, including, but not limited to, IL-alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.
In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), OPG, and neutrokine-alpha (International Publication No. WO 98/18921; OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 93/30693), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in Intentional Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/017832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5. FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINETM, PROKINETM), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGENTM), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGENTM, PROCRITTM), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of IL-1 through IL-12, interferon-gamma, or thrombopoietin.
In certain embodiments, albumin fusion proteins and/or polynucleotides of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.
In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).
In another embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na+-K+-2Cl− symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).
In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, 127I, radioactive isotopes of iodine such as 131I and 123I; recombinant growth hormone, such as HUMATROPETM (recombinant somatropin); growth hormone analogs such as PROTROPINTM (somatrem); dopamine agonists such as PARLODELTM (bromocriptine); somatostatin analogs such as SANDOSTATINTM (octreotide); gonadotropin preparations such as PREGNYLTM, A.P.L.TM and PROFASITM (chorionic gonadotropin (CG)), PERGONALTM (menotropins), and METRODINTM (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTRELTM and LUTREPULSETM (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRONTM (leuprolide acetate), SUPPRELINTM (histrelin acetate), SYNARELTM (nafarelin acetate), and ZOLADEXTM (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRHTM and THYPINONETM (protirelin); recombinant human TSH such as THYROGENTMsynthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T4TM, SYINTHROIDTM and LEVOTHROIDTM (levothyroxine sodium), L-T3TM, CYTOMELTM and TRIOSTATTM (liothyroine sodium), and THYROLARTM (liotx); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLETM (methimazole), NEO-MERCAZOLETM (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca2+ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUETM (iopanoic acid) and ORAGRAFINTM (sodium ipodate).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congagated estrogens such as ESTACETM (estradiol), ESTINYLTM (ethinyl estradiol), PREMARINTM, ESTRTABTM, ORTHO-ESTTM, OGENTM and estropipate (estrone), ESTROVISTM (quinestrol), ESTRADERMTM (estradiol), DELESTROGENTM and VALERGENTM (estradiol valerate), DEPO-ESTRADIOL CYPIONATETM and ESTROJECT LATM (estradiol cypionate); antiestrogens such as NOLVADEXTM (tamotifen), SEROPHENETM and CLOMIDTM (clomiphene); progestins such as DURALUTINTM (hydroxyprogesterone caproate), MPATM and DEPO-PROVERATM (medroxyprogesterone acetate), PROVERATM and CYCRINTM (MPA), MEGACETM (megestrol acetate), NORLUTINTM (norethindrone), and NORLUTATETM and AYGESTINTM (norethindrone acetate); progesterone implants such as NORPLANT SYSTEMTM (subdermal implants of norgestrel); antiprogestins such as RU 486TM (mifepristone); hormonal contraceptives such as ENOVIDTM (norethynodrel plus mestranol), PROGESTASERTTM (intrauterine device that releases progesterone), LOESTRINTM, BREVICONTM, MODICONTM, GENORATM, NELONATM, NORINYLTM, OVACON-35TM and OVACON-50TM (ethinyl estradiol/norethindrone), LEVLENTM, NORDETTETM, TRI-LEVLENTM and TRIPHASIL-21TM (ethinyl estradiol/levonorgestrel) LO/OVRALTM and OVRALTM (ethinyl estradiol/norgestrel), DEMULENTM (ethinyl estradiol/ethynodiol diacetate), NORINYLTM, ORTHO-NOVUMTM, NORETHINTM, GENOPRATM, and NELOVATM (norethindrone/mestranol), DESOGENTM and ORTHO-CEPTTM (ethinyl estradiol/desogestrel), ORTHO-CYCLENTM and ORTHO-TRICYCLENTM (ethinyl estradiol/norgestimate), MICRONORTM and NOR-QDTM (norethindrone), and OVRETTETM (norgestrel).
Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50TM (testosterone), TESTEXTM (testosterone propionate), DELATESTRYLTM (testosterone enanthate), DEPO-TESTOSTERONETM (testosterone cypionate), DANOCRINETM (danazol), HALOTESTINTM (fluoxymesterone), OREION METHYLTM, TESTREDTM and VIRILONTM (methyltestosterone), and OXANDRINTM (oxandrolone); testosterone transdermal systems such as TESODERMTM; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCURTM (cyproterone acetate), EULEXINTM (flutamide), and PROSCARTM (finasteride); adrenocorticotropic hormone preparations such as CORTROSYNTM (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATETM (alclometasone dipropionate), CYCLOCORTTM (amcinonide), BECLOVENTTM and VANCERILTM (beclomethasone dipropionate), CELESTONETM (betamethasone), BENISONETM and UTICORTTM (betamethasone benzoate), DEPROSONETM (betamethasone dipropionate), CELESTONE PHOSPHATETM (betamethasone sodium phosphate), CELESTONE SOLUSPANTM (betamethasone sodium phosphate and acetate), BETA-VALTM and VALISONETM (betamethasone valerate), TEMOVATETM (clobetasol propionate), CLODERMTM (clocortolone pivalate), CORTEFTM and HYDROCORTONETM (cortisol (hydrocortisone)), HYDROCORTONE ACETATETM (cortisol (hydrocortisone) acetate), LOCOIDTM (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATETM (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORTTM and SOLU CORTEFTM (cortisol (hydrocortisone) sodium succinate), WESTCORTTM (cortisol (hydrocortisone) valerate), CORTISONE ACETATETM (cortisone acetate), DESOWENTM and TRIDESILONTM (desonide), TOPICORTTM (desoximetasone), DECADRONTM (dexamethasone), DECADRON LATM (dexamethasone acetate), DECADRON PHOSPHATETM and HEXADROL PHOSPHATETM (dexamethasone sodium phosphate), FLORONETM and MAXIFLORTM (diflorasone diacetate), FLORINEF ACETATETM (fludrocortisone acetate), AEROBIDTM and NASALIDETM (flunisolide), FLUONIDTM and SYNALARTM (fluocinolone acetonide), LIDEXTM (fluocinonide), FLUOR-OPTM and FMLTM (fluorometholone), CORDRANTM (flurandrenolide), HALOGTM (halcinonide), HMS LIZUIFILMTM (medrysone), MEDROLTM (methylprednisolone), DEPO-MEDROLTM and MEDROL ACETATETM (methylprednisone acetate), A-METHAPREDTM and SOLUMEDROLTM (methylprednisolone sodium succinate), ELOCONTM (mometasone furoate), HALDRONETM (paramethasone acetate), DELTA-CORTEFTM (prednisolone), ECONOPREDTM (prednisolone acetate), HYDELTRASOLTM (prednisolone sodium phosphate), HYDELTRA-T.B.ATM (prednisolone tebutate), DELTASONETM (prednisone), ARISTOCORTTM and KENACORTTM (triamcinolone), KENALOGTM (triamcinolone acetonide), ARISTOCORTTM and KENACORT DIACETATETM (triamcinolone diacetate), and ARISTOSPANTM (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADRENTM (aminoglutethimide), NIZORALTM (ketoconazole), MODRASTANETM (trilostane), and METOPIRONETM (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULINTM and NOVOLINTM, oral hypoglycemic agents such as ORAMIDETM and ORINASETM (tolbu/tamide), DIABINESETM (chlorpropamide), TOLAMIDETM and TOLINASETM (tolazamide), DYMELORTM (acetohexamide), glibenclamide, MICRONASETM, DIBETATM and GLYNASETM (glyburide), GLUCUOROLTM (glipizide), and DIAMICRONTM (gliclazide), GLUCOPHAGETM (metformin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATINTM (octreotide); and diazoxides such as PROGLYCEMTM (diazoxide).
In one embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for uterine motility disorders. Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN® and ESTRATAB®), estradiols (e.g., CLIMARA® and ALORA®), estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate), PROMETRIUM® progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogens/medroxyprogesterone (e.g., PRMPROTM and PREMPHASE®) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRTTM).
In an additional embodiment, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromnic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOLTM), ferrous fumarate (e.g., FEOSTATTM), ferrous gluconate (e.g., FERGONTM), polysaccharide-iron complex (e.g., NIFEREXTM), iron dextran injection (e.g., INFEDTM), cupric sulfate, pyroxidine, riboflavin, Vitamin B12, cyancobalamin injection (e.g., REDISOLTM, RUBRAMIN PCTM), hydroxocobalamin, folic acid (e.g., FOLVITETM), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.
In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipraimine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), artianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).
In other embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).
In another embodiment, albumin fusion proteins and/or polynucleotides of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the albumin fusion proteins and/or polynucleotides of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.
In certain embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with treatments for gastrointestinal disorders. Treatments for gastrointestinal disorders that may be administered with the albumin fusion protein and/or polynucleotide of the invention include, but are not limited to, H2 histamine receptor antagonists (e.g., TAGAMETTM (cimetidine), ZANTACTM (ranitidine), PEPCIDTM (famotidine), and AXIDTM (nizatidine)); inhibitors of H+, K+ ATPase (e.g., PREVACIDTM (lansoprazole) and PRILOSECTM (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOLTM (bismuth subsalicylate) and DE-NOLTM (bismuth subcitrate)); various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTECTM (misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMOTILTM (diphenoxylate), MOTOFENTM (diphenoxin), and IMODIUMTM (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATINTM (octreotide), antiemetic agents (e.g., ZOFRANTM (ondansetron), KYTRILTM (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide and chlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.
In additional embodiments, the albumin fusion proteins and/or polynucleotides of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
The invention also provides a pharmaceutical pack or kit comprising, one or more containers filled with one or more of the ingredients of the pharmaceutical compositions comprising albumin fusion proteins of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Gene Therapy
Constructs encoding albumin fusion proteins of the invention can be used as a part of a gene therapy protocol to deliver therapeutically effective doses of the albumin fusion protein. A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, encoding an albumin fusion protein of the invention. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous nucleic acid molecules encoding albumin fusion proteins in vivo. These vectors provide efficient delivery of nucleic acids into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed “packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:27 1). A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988), Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., cited supra; Haj-Ahmand et al., J. Virol. 57:267 (1986)).
In another embodiment, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject nucleotide molecule by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. In a representative embodiment, a nucleic acid molecule encoding an albumin fusion protein of the invention can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992). No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
Gene delivery systems for a gene encoding an albumin fusion protein of the invention can be introduced into a patient by any of a number of methods. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific tansduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the, animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,323,470) or by Stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3 054-3 057). The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Where the albumin fusion protein can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the albumin fusion protein.
Additional Gene Therapy Methods
Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of an albumin fusion protein of the invention. This method requires a polynucleotide which codes for an albumin fusion protein of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the fusion protein by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the fusion protein of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which ate engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
In one embodiment, polynucleotides encoding the albumin fusion proteins of the present invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, polynucleotides encoding the albumin fusion proteins of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and PSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters: the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the gene corresponding to the Therapeutic protein portion of the albumin fusion proteins of the invention.
Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypepide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney; gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg, to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the tradermark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., PCT Publication No. WO 90/11097 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.) or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DPPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucteopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et at., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA. 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittrmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the radon will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859; 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-caflonic lipid complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding an albumin fusion protein of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avianleukosis virus, gibbon ape leukemia virus; human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding an albumin fusion protein of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a fusion protin of the present invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses fusion protein of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis. 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).
Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be crown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
Preferably, the adenoviruses used in the present invention are replication deficient Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Nluzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,673, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a fsuion protein of the invention.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide, sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijistra et al., Nature 342:435-438 (1989), which are herein encorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.
The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
The polynucleotide encoding an albumin fusion protein of the present invention may contain a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
A preferred method of local administration is by direct injection. Preferably, an albumin fusion protein of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
Therapeutic compositions useful in systemic administration, include fusion proteins of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising albumin fusion proteins of the invention for targeting the vehicle to a particular site.
Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the out of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian
Albumin fusion proteins of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, nice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
Biological Activities
Albumin fusion proteins and/or polynucleotides encoding albumin fusion proteins of the present invention, can be used in assays to test for one or more biological activities. If an albumin fusion protein and/or polynucleotide exhibits an activity in a particular assay, it is likely that the Therapeutic protein corresponding to the fusion portein may be involved in the diseases associated with the biological activity. Thus, the fusion protein could be used to treat the associated disease.
Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, albumin fusion proteins of the invention and polynucleotides encoding these proteins, may be used in diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with aberrant activity of secreted polypeptides.
In preferred embodiments, fusion proteins of the present invention may be used in the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders relating to diseases and disorders of the endocrine system, the nervous system (See, for example, “Neurological Disorders” section below), the immune system (See, for example, “Immune Activity” section below), respiratory system (See, for example, “Respiratory Disorders” section below), cardiovascular system (See, for example, “Cardiovascular Disorders” section below), reproductive system (See, for example, “Reproductive System Disorders” section below) digestive system (See, for example, “Gastrointestinal Disorders” section below), diseases and/or disorders relatin, to cell proliferation (See, for example, “Hyperproliferative Disorders/Cancer” section below), and/or diseases or disorders relating to the blood ((See, for example, “Blood-Related Disorders” section below).
In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indication Y” column of Table 1 comprising administering to a patient in which such treatment, prevention or amelioration is desired an albumin fusion protein of the invention that comprises a Therapeutic protein portion corresponding to a Therapeutic protein disclosed in the “Therapeutic Protein X” column of Table 1 (in the same row as the disease or disorder to be treated is listed in the “Preferred Indication Y” column of Table 1) in an amount effective to treat, prevent or ameliorate the disease or disorder.
In certain embodiments, an albumin fusion protein of the present invention may be used to diagnose and/or prognose diseases and/or disorders associated with the tissue(s) in which the gene corresponding to the Therapeutic protein portion of the fusion portien of the invention is expressed.
Thus, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.
More generally, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful for the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with the following systems.
Immune Activity
Albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophaaes) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as a marker or detector of a particular immune system disease or disorder.
In another embodiment, a fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies. Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypoeammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IcM, IgG and IgA deficiency with increased IgM; antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.
In specific embodiments, ataxia-telangiectasia or conditions associated with atakia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeore anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant. T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose phosphate dehydroenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thynic alyipphoplasia-aplasia immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In a preferred embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, one or more of the following systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytgpenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
Additional disorders that are likely to have an autoimmune component that may be treated prevented, and/or diagnosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes meuitus, and autoimmune inflammatory eye disorders.
Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermiatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone AINA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urdcaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized; e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgVI antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.
In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using fusion, proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a immunosuppressive agent(s).
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, prognosing, and/or diagnosing, diseases, disorders, and/or conditions of hematopoietic cells. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types heratopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Moreover, these molecules can be used to treat, prevent, proganose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate IgE concentrations in vitro or in vivo.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).
Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, have uses in the treatment of issue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, eadocarditis, esophagits, eustachiris, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retnitis, rhinitis, salpingitis, scieritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.
In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. Oran rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
In other embodiments, fusion proteins of the invention and/or polynucleotides encodin albumin fusion proteins of the invention, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance tumor-specific immune responses.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of. AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HFV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the croup consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi. 
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
In one embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are administered to an animal (e.g., mouse, rat, rabbit hamster, guinea pig, pigs, micro-pig, chicken, camel, coat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell responsiveness to pathogens.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an activator of T cells.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to induce higher affinity antibodies.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to increase serum immunoglobulin concentrations.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to accelerate recovery of immunocompromised individuals.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an adjuvant to boost immunoresponsiveness among aged populations and/or neonates.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as an agent to direct an individual's immune system towards development of a humnoral response (i.e. TH2) as opposed to a TH1 cellular response.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplasic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in the pretreatment of bone marrow samples prior to transplant.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence/immunodeficiency such as observed among SCID patients.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
In another specific embodiment albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit complement mediated cell lysis.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be employed to treat adult respiratory distress syndrome (ARDS).
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to stimulate the regeneration of mucosal surfaces.
In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, ocitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease (“CVID”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.
In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to diagnose, pronose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled “Hyperproliferative Disorders” elsewhere herein.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a therapy for decreasing cellular proliferation of Large Bell Lymphomas.
In another specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has underdone a partial or complete splenectomy.
Blood-related Disorders
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.
In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed.
The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to modulate hem atopoietic activity (the formation of blood cells). For example, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of anemias and leukopenias described below. Alternatively, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.
The fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to prevent, treat, or diagnose blood dyscrasia.
Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias. Anemias that may be treated prevented or diagnosed by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary sideroblastic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa dapsone, and/or sulfadrugs. Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose phosphate dehydrogenase deficiency, and sickle cell anemia.
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease). Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating thalassemias, including, but not limited to, major and minor forms of alpha-thalassemia and beta-thalassemia.
In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing preventing, and/or treating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor LX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber, syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
The effect of the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.
Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.
In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukopenia. In other specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.
Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticoagulant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felry syndrome, malaria and sarcoidosis), and some drug treatment regimens.
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited to, lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskort-Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing and/or treating diseases and disorders associated with macrophages numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.
In another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing; preventing, and/or treating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.
In yet another embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias. T cell leukemias, Sezary syndrome, and Hairy cell leukenia), chronic myelocytic (myeloid, myelogendus, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodk in's lymphoma. Burkitt's lymphoma, and mycosis fungoides.
In other embodiments, the albumin fusion, proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies., monoclonal gammopathies of undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating, preventing, and/or diagnosing myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as a treatment prior to surgery, to increase blood cell production.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as an agent to increase cytokine production.
In other embodiments, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.
Hyperproliferative Disorders
In certain embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing, an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract
Similarly, other hyperproliferative disorders can also be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute. Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumorsi Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Exrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodakin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferaive Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squarhous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheocbromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology 2d Ed., W. B. Saunders Co., Philadelphia, pp. 63-79.)
Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Hyperplatic disorders which can be diagnosed, prognosed; prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include; but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endothelial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatois hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.
Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephaloophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoacbondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
Additional pre-neoplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose and/or prognose disorders associated with the tissue(s) in which the polypeptide of the invention is expressed.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to, those described herein. In a further preferred embodiment albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.
Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis. For example, diseases associated with increased cell survival or the inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides polypeptides and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyneioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include AIDS, neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., bepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
Hyperproliferative diseases and/or disorders that could be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis skin, soft tissue, spleen, thorax, and urogenital tract.
Similarly, other hyperproliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoiddsis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
Another preferred embodiment utilizes polynucleotides encoding albumin fusion proteins of the invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide encoding an albumin fusion protein of the present invention, wherein said polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing, a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the fusion protein of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene trascript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:312 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference).
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. These fusion protieins and/or polynucleotides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, these fusion proteins and/or polynucleotides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of these proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses. 50(5):423-33 (1998) Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Ned. 76(6):402-12 (1998), Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporated by reference).
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering these albumin fusion proteins and/or polynucleotides, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
In another embodiment, the invention provides a method of delivering compositions containing the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to targeted cells expressing the a polypeptide bound by, that binds to, or associates with an albumin fuison protein of the invention. Albumin fusion proteins of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
Albumin fusion proteins of the invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the albumin fusion proteins of the invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
Renal Disorders
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferaive glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolitbiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.)
In addition, compositions of the invention can be used to diagnose, prognose, prevent, and/or treat metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).
Compositions of the invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypemephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephrids, proteinuria, hyponatremia, hypematremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
Compositions of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Compositions of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.
Cardiovascular Disorders
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, dianose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Elsenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outdet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischermia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postixricardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, exttrasystole, Adamstokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, artertis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telanaiectasia, atacia telangiecmasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiltis obliterans.
Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and hrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Methods of delivering polynucleotides are described in more detail herein.
Respiratory Disorders
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders of the respiratory system.
Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngiris (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wegener's granulomatosis (ranulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).
Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.; candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydia spp.), opporrunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothora, (e., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe disease, Hand-Schuller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.
Anti-angiogenesis Activity
The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth; these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1935); Patz, Am. J. Opthalmol. 94:715-743 (1982): and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to solid tumors, including prostates lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer, primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be delivered topically, in order to treat cancers such as skin cancer, head and heck tumors, breast tumors, and Kaposi's sarcoma.
Within yet other aspects, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel Growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculooenesis; Granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to a hypertrophic scar or keloid.
Within one embodiment of the present invention fusion proteins, of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are directly injected into-a hypercrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., bums), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
Moreover, Ocular disorders associated with neovascularization which can be treated with the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (e.g., fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the, cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patients visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states and as a complication of wearing contact lenses.
Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections night only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eyes, such that the formation of blood vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoaculation.
Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of an albumin fusion protein of the invention and/or polynucleotides encoding an albumin fusion protein of the invention to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.
Additionally, disorders which can be treated with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be treated, prevented, diagnosed, and/or prognosed with the the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention of the invention include, but-are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyouenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration; corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.
In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be incorporated into surgical sutures in order to prevent stitch granulomas.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a survical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising, administering albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the and-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of the present invention, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Acivator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H) oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin. (Tomkinson et al., Biochem J. 286:475-430, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4): 1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, (1992)); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, diagnosed, and/or prognosed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox. viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
In preferred embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be treated or detected by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, progression, and/or memastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (gnulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, prevented, diagnosed, and/or prognesed using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthitis) myclodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, bums resulting from beat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote dermal reestablishment subsequent to dermal loss.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to increase adherence to a wound bed: autogralts, artificial skin, allografts, autodermic raft, autoepdermic grafts, avacular rafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar oraft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to promote skin strength and to improve the appearance of aged skin.
It is believed that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to reduce the side effects of out toxicity that result from radiation, chemotherapy treatments or viral infections. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may have a cytoprotective effect on the small intestine mucosa. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous lands), treatment of other skin defects such as psoriasis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Albumin fusion proteins of the invention and/or polynucleotides encodin, albumin fusion proteins of the invention, could be used to treat diseases associate with the under expression.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaininophen, carbon tetraholoride and other hepatotoxins known in the art).
In addition, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
Neural Activity and Neurological Diseases
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency; folic acid deficiency, Wemicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited-to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
In one embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of. the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.
In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.
In another preferred embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack
The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci., 4:17-42 (1981); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Miotorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Further, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may play a role in neuronal survival; synapse formation; conductance; neural differentiation, etc. Thus, compositions of the invention (including fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention) may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders. The compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders. Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.
Additionally, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral. ischemia, Subclayian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used to treat and/or detect neurologic diseases. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.
Examples of neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encodin, albumin fusion proteins of the invention include, brain diseases; such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neopasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central nervous system infections such as AIS Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyeitis, Visna, and cerebral malaria.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include meningitis such as arachnoiditis, asepic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meninococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal. Meningitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uveomeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as amyptonia congenita amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-Biedi Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube defects such as anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations (such as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development, disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wemicke Aphasia, articulation disorders, communicative disorders such as speech disorders which include dysarthria, echolalia, mutism and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff-man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressive external ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia, such as Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders such as hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as amyotonia congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adie's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome, Chronic Progressive External Ophthalmoplecia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demiyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as diabetic foot.
Additional neurologic diseases which can be treated or detected with fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome neuralgia such as causalgia, cervicabrachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
Endocrine Disorders
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.
Hormones secreted by the lands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions many be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).
Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma—islet cell tumor syndrome;, disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease; Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.
In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
In another embodiment, albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders associated with the tissue(s) in which the Therapeutic protein corresponding to the Therapeutic protein portion of the albumin protein of the invention is expressed,
Reproductive System Disorders
The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used for the diagnosis, treatment, or prevention of diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermnia, azoospermia, oligospermia, and teratozoospermia).
Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
Additionally, the compositions of the invention may be useful in the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbornembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.
Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
Other disorders and/or diseases of the male reproductive system include, for example, Kinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
Further, the polynucleotides, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen scierosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaoinitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia
Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicomuate uterus, unicdmuare uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.
Ovarian diseases and/or disorders include anovulation, polycysric ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestatonal diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. Additionally, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders, and obstruction of the intestine.
Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
Other disorders and/or diseases of the female reproductive system that may be diaonosed, treated, and/or prevented by the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.
Infectious Disease
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papilora virus, Papovaviridae, Parvoviridae. Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat patients nonresponsive to one or more other commercially available he patitis vaccines. In a further specific embodiment fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat AIDS.
Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but not limited to, the following Gram-Negative and Gram-positive bacteria, bacterial families, and fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Asperoillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chiamydia, Clostndium (e.g., Closmidium borulinum, Closrridium dificile, Clostridium perfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g., Corynebacrerium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorragic E. coli), Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella ryphi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospiral Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibro (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections, noscomial infections. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat: tetanus, diptheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schisisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malarie and Plasmodiun ovale). These parasites can cause a variety of diseases or symptoms, including, but not, limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention are used to treat, prevent, and/or diagnose malaria.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could either be by administering an effective amount of an albumin fusion protein of the invention to the patient, or by removing cells from the patient, supplying the, cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
Regeneration
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
Moreover, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma cerebrovascular disease, and stoke). Specifically, diseases-associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Drager syndrome), could all be treated using the albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention.
Gastrointestinal Disorders
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention, may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and structuring, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, astritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritoneal diseases (e.g., chyloperioneurm, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).
Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndromre, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphylloborhrium spp., and T. solium).
Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchymal, hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenomia), Hepatocyte(Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).
Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the Gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung, disease, toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery); duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis. (enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis), immnunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula.(rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric, vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric oulet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis ravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.
Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).
Chemotaxis
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
Albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may increase chemotaxic activity of particular cells. These chemotacic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention could be used as an inhibitor of chemotaxis.
Binding Activity
Albumin fusion proteins of the invention may be used to screen for molecules that bind to the Therapeutic protein portion of the fusion protein or for molecules to which the Therapeutic protein portion of the fusion protein binds. The binding of the fusion protein and the molecule may activate (agonist), increase, inhibit (antagonist); or decrease activity of the fusion protein or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the Therapeutic protein portion of the fusion protein of the invention, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the Therapeutic protein portion of an albumin fusion protein of the invention binds, or at least, a fragment of the receptor capable of being bound by the Therapeutic protein portion of an albumin fusion protein of the invention (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate cells which express the albumin fusion proteins of the invention. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. 
The assay may simply test binding of a candidate compound to an albumin fusion protein of the invention, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the fusion protein.
Alternatively, the assay can be carried out using cell-free preparations, fusion protein/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing an albumin fusion protein, measuring fusion protein/molecule activity or binding, and comparing the fusion protein/molecule activity or binding to a standard.
Preferably, an ELISA assay can measure fusion protein level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure fusion protein level or activity by either binding, directly or indirectly, to the albumin fusion protein or by competing with the albumin fusion protein for a substrate.
Additionally, the receptor to which a Therapeutic protein portion of an albumin fusion protein of the invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, in cases wherein the Therapeutic protein portion of the fusion protein corresponds to FGF, expression cloning may be employed wherein polyadenylated RNA is prepared from a cell responsive to the albumin fusion protein, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the albumin fusion protein. Transfected cells which are grown on glass slides are exposed to the albumin fusion protein of the present invention, after they have been labeled. The albumin fusion proteins can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, a labeled albumin fusion protein can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule for the Therapeutic protein component of an albumin fusion protein of the invention, the linked material may be resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the fusion protein can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of the fusion protein, and/or Therapeutic protein portion or albumin component of an albumin fusion protein of the present invention, thereby effectively generating agonists and antagonists of an albumin fusion protein of the present invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 237:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides encoding albumin fusion proteins of the invention and thus, the albumin fusion proteins encoded thereby, may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific recombination. In another embodiment, polynucleotides encoding albumin fusion proteins of the invention and thus, the albumin fusion proteins encoded thereby, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of an albumin fusion protein of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BLMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inbibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the Therapeutic protein portion arid/or albumin component of the albumin fusion proteins of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a Therapeutic protein portion and/or albumin component of the albumin fusion proteins of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
Additionally, this invention provides a method of screening compounds to identify those which modulate the action of an albumin fusion protein of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, an albumin fusion protein of the present invention, and the compound to be screened and 3[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates-proliferation by determining the uptake of 3[H] thymidine in-each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
In another method, a mammalian cell or membrane preparation expressing a receptor for the Therapeutic protien component of a fusion protine of the invention is incubated with a labeled fusion protein of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential fusion protein. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the fusion protein/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the albumin fusion proteins of the invention from suitably manipulated cells or tissues.
Therefore, the invention includes a; method of identifying compounds which bind to an albumin fusion protein of the invention comprising the steps of: (a) incubating a candidate binding compound with an albumin fusion protein of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonist/antagonists comprising the steps of: (a) incubating a candidate compound with an albumin fusion protein of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the fusion protein has been altered.
Targeted Delivery
In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a component of an albumin fusion protein of the invention.
As discussed herein, fusion proteins of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering fusion proteins of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a Therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering an albumin fusion protein of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricing abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
Drug Screening
Further contemplated is the use of the albumin fusion proteins of the present invention, or the polynucleotides encoding these fusion proteins, to screen for molecules which modify the activities of the albumin fusion protein of the present invention or proteins corresponding to the Therapeutic protein portion of the albumin fusion protein. Such a method would include contacting the fusion protein with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of the fusion protein following binding.
This invention is particularly useful for screening therapeutic compounds by using the albumin fusion proteins of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The albumin fusion protein employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotc host cells which are stably transformed with recombinant nucleic acids expressing the albumin fusion protein. Drugs are screened against such transformed cells or supernatants obtained from culturing such cells, in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and an albumin fusion protein of the present invention.
Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the albumin fusion proteins of the present invention. These methods comprise contacting such an agent with an albumin fusion protein of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the albumin fusion protein or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the albumin fusion protein of the present invention.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to an albumin fusion protein of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1904, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with an albumin fusion protein of the present invention and washed. Bound peptides are then detected by methods well known in the art. Purified albumin fusion protein may be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding an albumin fusion protein of the present invention specifically compete with a test compound for binding to the albumin fusion protein or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with an albumin fusion protein of the invention.
Binding Peptides and Other Molecules
The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind albumin fusion proteins of the invention, and the binding molecules identified thereby These binding molecules are useful, for example, as agonists and antagonists of the albumin fusion proteins of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:

    
    
        contacting an albumin fusion protein of the invention with a plurality of molecules; and
        identifying a molecule that binds the albumin fusion protein.
    
    


The step of contacting the albumin fusion protein of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the albumin fusion protein on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized albumin fusion protein of the invention. The molecules having a selective affinity for the albumin fusion protein can then be purified by affinity selection. The nature of the solid support, process for attachment of the albumin fusion protein to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by an albumin fusion protein of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the albumin fusion protein and the individual clone. Prior to contacting the albumin fusion protein with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for an albumin fusion protein of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for an albumin fusion protein of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of an albumin fusion protein of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the albumin fusion protein of the invention or the plurality of polypeptides are bound to a solid support
The plurality of molecules provided according, to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind an albumin fusion protein of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., Science 251:767-773 (1991); Houghten et al., Nature 354:84-86 (1991); Lam et al., Nature 354:82-84 (1991); Medynski, Bio/Technology 12:709-710 (1994); Gallop et al., J. Medicinal Chemistry 37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993); Erb et al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994); Houahten et al., Biotechniques 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA 91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA 90:11708-11712 (1993); PCT Publication No. WO 93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992).
Examples of phage display libraries are described in Scott et al;, Science 249:386-390 (1990); Devlin et al., Science, 249-4046 (1990); Christian et al., 1992, J. Mol. Biol. 227:711-718 1992); Lenstra, J. Immunol. Meth. 152:149-157 (1992); Kay et al, Gene 128:59-65 (1993); and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-9026 (1994).
By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994)) can be adapted for use. Peptoid libraries (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992)) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natl. Acad. Sci. USA 91:11138-11142 (1994)).
The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke (Bio/Technology 13:351-360 (1995) list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xantbines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.
Non-peptide libraries can be classified broadly into two types: decorated monomers and oiligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.
Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley et al., Adv. Exp. Med. Biol. 251:215-218 (1989); Scott et al. Science 249:386-390 (1990); Fowlkes et al., BioTechniques 13:422-427 (1992); Oldenburg, et al., Proc. Natl. Acad. Sci. USA 89:5393-5397 (1992); Yu et al., Cell 76:933-945 (1994); Staudt et al., Science 241:577-580 (1988); Bock et al., Nature 355:564-566 (1992); Tuerk et al., Proc. Natl. Acad. Sci. USA 89:6988-6992 (1992); Ellington et al., Nature 355:850-852 (1992); U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al., Rebar et al., Science 263:671-673 (1993); and PCT Publication No. WO 94/18318.
In a specific embodiment, screening, to identify a molecule that binds an albumin fusion protein of the invention can be carried out by contacting the library members with an albumin fusion protein of the invention immobilized on a solid phase and harvesting those library members that bind to the albumin fusion protein. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley et al., Gene 73:305-318 (1983); Fowlkes et al., BioTechniques 13:422-427 (1992); PCT Publication No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields et al., Nature 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991) can be used to identify molecules, that specifically bind to polypeptides of the invention.
Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.
As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment a binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.
The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.
Other Activities
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. The albumin fusion proteins of the invention and/or polynucleotides encoding albumin fusion proteins of the invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for treating wounds due to injuries, burns, postoperative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease and AIDS-related complex. An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along, the same lines, an albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, an albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by. Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
An albumin fusion protein of the invention and/or polynucleotide encoding an albumin fusion protein of the invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, oat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, oat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the alterations detected in the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
EXAMPLES
Example 1
Preparation of HA-hGH Fusion Proteins
An HA-hGH fusion protein was prepared as follows:
Cloning of hGH cDNA
The hGH cDNA was obtained from a human pituitary gland cDNA library (catalogue number HL1097v, Contech Laboratories, Inc) by PCR amplification. Two oligonucleotides suitable for PCR amplification of the hGH cDNA, HGH1 and HGH2, were synthesized using an Applied Biosystems 380B Oligonucleotide Synthesizer.

    
    
        HGH1: 5′-CCCAAGAATTCCCTTATCCAGGC-3′ (SEQ ID NO: 1)
        HGH2: 5′-GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG-3′ (SEQ ID NO: 2)
    
    


HGH 1 and HGH2 differed from the equivalent portion of the hGH cDNA sequence (Martial et al., 1979) by two and three nucleotides, respectively, such that after PCR amplification an EcoRI site would be introduced to the 5′ end of the cDNA and a BamHI site would be introduced into the 3′ end of the cDNA. In addition, HGH2 contained a HindIII site immediately downstream of the hGH sequence.
PCR amplification using a Perkin-Elmer-Cetus Thermal Cycler 9600 and a Perkin-Elmer-Cetus PCR kit, was performed using single-stranded DNA template isolated from the phage particles of the cDNA library as follows: 10 μL phage particles were lysed by the addition of 10 μL phage lysis buffer (280 μg/mL proteinase K in TE buffer) and incubation at 55° C. for 15 min followed by 85° C. for 15 min. After a 1 min. incubation on ice, phage debris was pelleted by centrifugation at 14,000 rpm for 3 min. The PCR mixture contained 6 μL of this DNA template, 0.1 μM of each primer and 200 μM of each deoxyribonucleotide. PCR was carried out for 30 cycles, denaturing at 94° C. for 30 s, annealing at 65° C. for 30 s and extending at 72° C. for 30 s, increasing the extension time by 1 s per cycle.
Analysis of the reaction by gel electrophoresis showed a single product of the expected size (589 base pairs).
The PCR product was purified using Wizard PCR Preps DNA Purification System (Promega Corp) and then digested with EcoRI and HindIII. After further purification of the EcoRI-HindIII fragment by gel electrophoresis, the product was cloned into pUC19 (GIBCO BRL) digested with EcoRI and HindIII, to give pHGH1. DNA sequencing of the EcoRI HindIII region showed that the PCR product was identical in sequence to the hGH sequence (Marial et al., 1979), except at the 5′ and 3′ ends, where the EcoRI and BamHI sites had been introduced, respectively.
Expression of the hGH cDNA.
The polylinker sequence of the phagemid pBluescribe (+) (Stratagene) was replaced by inserting an oligonucleotide linker, formed by annealing two 75-mer oligonucleotides, between the EcoRI and HindIII sites to form pBST(+). The new polylinker included a unique NotI site.
The NotI HA expression cassette of pAYE309 (EP 431 880) comprising the PRBI promoter, DNA encoding the HA/MFα-1 hybrid leader sequence, DNA encoding HA, and the ADH1 terminator, was transferred to pBST(+) to form pHA1. The HA coding sequence was removed from this plasmid by digestion with HindIII followed by religation to form pHA2.
Cloning of the hGH cDNA, as described in Example 1, provided the hGH coding region lacking the pro-hGH sequence and the first 8 base pairs (bp) of the mature hGH sequence. In order to construct an expression plasmid for secretion of hGH from yeast, a yeast promoter, signal peptide and the first 8 bp of the hGH sequence were attached to the 5′ end of the cloned hGH sequence as follows: The HindIII-SfaNI fragment from pRA 1 was attached to the 5′ end of the EcoRI/HindIII fragment from pHGHI via two synthetic oligonucleotides, HGH3 and HGH4 (which can anneal to one another in such a way as to generate a double stranded fragment of DNA with sticky ends that can anneal with SfaNIand EcoRI sticky ends):

    
    
        HGH3: 5′-GATAAAGATTCCCAAC-3′ (SEQ ID NO: 3)
        HGH4: 5′-AATTGTTGGGAATCTTT-3′ (SEQ ID NO: 4)
    
    


The HindIII fragment so formed was cloned into HindIII-digested pHA2 to make pHGH2, such that the hGH cDNA was positioned downstream of the PRBI promoter and HA/MFα-1 fusion leader sequence (see, e.g., International Publication No. WO90/01063). The NotI expression cassette contained in pHGH2, which included the ADH1 terminator downstream of the hGH cDNA, was cloned into NotI-digested pSAC35 (Sleep et al., BioTechnology 8:42 (1990)) to make pHGH12. This plasmid comprised the entire 2 μm plasmid to provide replication functions and the LEU2 gene for selection of transformants
pHGH12 was introduced into S. cerevisiae D88 by transformation and individual transformants were grown for 3 days at 30° C. in 10 mL YEPD (1% w/v yeast extract, 2% w/v, peptone, 2% w/v, dextrose).
After centrifugation of the cells, the supernatants were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were found to contain protein which was of the expected size and which was recognized by anti-hGH antiserum (Sigma, Poole, UK) on Western blots.
Cloning and Expression of an HA-hGH Fusion Protein.
In order to fuse the HA cDNA to the 5′ end of the hGH cDNA, the pHA1 HindIII-Bsu36I fragment (containing most of the HA cDNA) was joined to the pHGH1 EcoRI-HindIII fragment (containing most of the hG4 cDNA) via two oligonucleotides, HGH7 and HGH8

    
    
        HGH7: 5′-TTAGGCTTATTCCCAAC 3′ (SEQ ID NO: 5)
        HGH8: 5′-AATTGTTGGGAATAAGCC 3′ (SEQ ID NO: 6)
    
    


The HindIII fragment so formed was cloned into pHA2 digested with HindIII to make pHGH10, and the NotI expression cassette of this plasmid was cloned into NotI-digested pSAC35 to make pHGH16.
pHGH 16 was used to transform S. cerevisiae D88 and supernatants of cultures were analyzed as described above. A predominant band was observed that had a molecular weight of approximately 88 kD, corresponding to the combined masses of HA and hGH. Western blotting using anti-HA and anti-hGH antisera (Sigma) confirmed the presence of the two constituent parts of the albumin fusion protein.
The albumin fusion protein was purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography. Analysis of the N-terminus of the protein by amino acid sequencing confirmed the presence of the expected albumin sequence.
An in vitro growth hormone activity assay (Ealey et al., Growth Regulation 5:36 (1995)) indicated that the albumin fusion protein possessed full hGH activity. In a hypophysectomised rat weight gain model, performed essentially as described in the European Pharmacopoeia (1987, monograph 556), the fusion molecule was more potent than hGH when the same number of units of activity (based on the above in vitro assay) were administered daily. Further experiments in which the albumin fusion protein was administered once every four days showed a similar overall growth response to a daily administration of hGH. Pharmacokinetic experiments in which 125I-labeled protein was administered to rats indicated an approximately ten-fold increase in circulatory half-life for the albumin fusion protein compared to hGH.
A similar plasmid was constructed in which DNA encoding the S. cerevisiae invertase (SUC2) leader sequence replaced the sequence for the hybrid leader, such that the encoded leader and the junction (↓) with the HA sequence were as follows:

    
    
        . . . MLLQAFLFLLAGFAAKISA↓DAHKS . . . (SEQ ID NO: 7) Invertase leader HA sequence . . .
    
    


On introduction into S. cerevisiae DB1, this plasmid directed the expression and secretion of the albumin fusion protein at a level similar to that obtained with pHGH16. Analysis of the N-terminus of the albumin fusion protein indicated precise and efficient cleavage of the leader sequence from the mature protein.
Cloning and Expression of an hGH-HA Fusion Protein.
In order to fuse the hGH cDNA to the 5′ end of the HA cDNA, the HA cDNA was first altered by site-directed mutagenesis to introduce an EcoNI site near the 5′ end of the coding region. This was done by the method of Kunkel et al. (Methods in Enzymol. 154:367 (1987)) using single-stranded DNA template prepared from pHAI and a synthetic oligonucleotide, LEU4:

    
    
        LEU4: 5′-GAGATGCACACCTGAGTGAGG-3′ (SEQ ID NO: 8)
    
    


Site-directed mutagenesis using this oligonucleotide changed the coding sequence of the HA cDNA from Lys4 to Leu4 (K4L). However, this chance was repaired when the hGH cDNA was subsequently joined at the 5′ end by linking the pHGH2 NotI-BamHI fragment to the EcoNI-NotI fragment of the mutated pHAI, via the two oligonucleotides HGH5 and HGH6:

    
    
        HGH5: 5′-GATCCTGTGGCTTCGATGCACACAAGA-3′ (SEQ ID NO: 9)
        HGH6: 5′-C7CTTGTGTGCATCGAAGCCACAG-3′ (SEQ ID NO: 10)
    
    


The NotI fragment so formed was cloned into NotI-digested pSAC35 to make pHGH14. pHGH14 was used to transform S. cerevisiae D88 and supernatants of culture were analyzed as above. A predominant band was observed that had a molecular weight of approximately 88 kD, corresponding to the combined masses of hGH and HA. Western blotting using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.
The albumin fusion protein was purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography. Analysis of the N-terminus of the protein by amino acid sequencing confirmed the presence of the expected hGH sequence.
In vitro studies showed that the albumin fusion protein retained hGH activity, but was significantly less potent than an albumin fusion protein comprising full length HA (1-585) as the N-terminal portion and hGH as the C-terminal portion, as described above.
Construction of Plasmids for the Expression of hGH Fusions to Domains of HA.
Fusion polypeptides were made in which the hGH molecule was fused to the first two domains of HA (residues 1 to 387). Fusion to the N terminus of hGH was achieved by joining the pHA1 HindIII-SapI fragment, which contained most of the coding sequence for domains 1 and 2 of HA, to the pHGHI EcoRI-HindIII fragment, via the oligonucleotides HGH 11 and HGH 12:

    
    
        HGH11: 5′-TGTGGAAGAGCCTCAGAATTTATCCCAAC-3′ (SEQ ID NO: 11)
        HGH12: 5′-AATTGTGGGAATAAATTCTGAGGCTCTTCC-3′ (SEQ ID NO: 12)
    
    


The HindIII fragment so formed was cloned into HindIII-digested pHA2 to make pHGH37 and the NotI expression cassette of this plasmid was cloned into. NotI-digested pSAC35.
The resulting plasmid, pHGH38, contained an expression cassette that was found to direct secretion of the fusion polypeptide into the supernatant when transformed into S. cerevisiae DB 1. Western blotting using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.
The albumin fusion protein was purified from culture supernatant by cation exchange chromatography followed by gel permeation chromatography.
In vivo studies with purified protein indicated that the circulatory half-life was longer than that of hGH, and similar to that of an albumin fusion protein comprising full-length HA (1-535) as the N-terminal portion and hGH as the C-terminal portion, as described above. In vitro studies showed that the albumin fusion protein retained hGH activity,
Using a similar strategy as detailed above, an albumin fusion protein comprising the first domain of HA (residues 1-194) as the N-terminal portion and hGH as the C-terminal portion, was cloned and expressed in S. cerevisiae DBL. Western blotting of culture supernatant using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion protein.
Fusion of HA to hGH Using a Flexible Linker Sequence
Flexible linkers, comprising repeating units of [Gly-Gly-Gly-Gly-Ser]n, where n was either 2 or 3, were introduced between the HA and hGH albumin fusion protein by clonin of the oligonucleotides HGH16, HGH17, HGH18 and HGH19:

    
    
        HGH16:5′-TTAGGCTTAGGTGGCGGTGGATCCGGCGGTGGTGGATCTTTCCCA AC-3′ (SEQ ID NO: 13)
        HGH17:5′-AATTGTTGGGAAAGATCCACCACCGCCGGATCCACCGCCACCTAAGCC-3′ (SEQ ID NO: 14)
        HGH18:5′-TTAGGCTTAGGCGGTGGTGGATCTGGTGGCGGCGGATCTGGTGGCGGTGGATCCTTCCCAAC-3′ (SEQ ID NO: 15).
        HGH19:5′-AATTGTTGGGAAGGATCCACCGCCACCAGATCCGCCGCCACCAGATCCACCACCGCCTAAGCC-3′ (SEQ ID NO: 16)
    
    


Annealing of HGH16 with HGH17 resulted in n=2, while HGH18 annealed to HGH19 resulted in n=3. After annealing, the double-stranded oligonucleotides were cloned with the EcoRI-Bsu361 fragment isolated from pHGH1 into Bsu361-divested pHGH10 to make pHGH56 (where n=2) and pHGH57 (where n=3). The NotI expression cassettes from these plasmids were cloned into NotI-digested pSAC35 to make pHGH58 and pHOH59, respectively.
Cloning of the oligonucleotides to make pHGH56 and pHGH57 introduced a BamHI site in the linker sequences. It was therefore possible to construct linker sequences in which n=1 and n=4, by joining either the HindIII-BamHI fragment from pHGH56 to the BamHI-HindIII fragment from pHGH57 (making n=1), or the HindIII-BamHI fragment from pHGH57 to the BamHI-HindIII fragment from pHGH56 (making n=2). Cloning of these fragments into the HindIII site of pHA2, resulted in pHGH60 (n=1) and pHGH61 (n=4). The NotI expression cassettes from pHGH60 and pHGH61 were cloned into NotI-digested pSAC35 to make pHGH62 and pHGH63, respectively.
Transformation of S. cerevisiae with pHGH58, pHGH59, pHGH62 and pHGH63 resulted in transformants that secreted the fusion polypeptides into the supernatant. Western blotting using anti-HA and anti-hGH antisera confirmed the presence of the two constituent parts of the albumin fusion proteins.
The albumin fusion proteins were purified from culture supernatant by cation exchange chromatography, followed by anion exchange and gel permeation chromatography. Analysis of the N-termini of the proteins by amino acid sequencing confirmed the presence of the expected albumin sequence. Analysis of the purified proteins by electrospray mass spectrometry confirmed an increase in mass of 315 D (n=1), 630 D (n=2), 945 D (n=3) and 1260 D (n=4) compared to the HA-hGH fusion protein described above. The purified protein was found to be active in vitro.
Increased Shelf-Life of HA-hGH Fusion Proteins: Methods
HA-hGH and hGH were separately diluted in cell culture media containing 5% horse serum to final concentrations of 100-200 μg/ml and incubated at 4, 37 or 50° C. At time zero and at weekly intervals thereafter, aliquots of the samples were tested for their biological activity in the Nb2 cell proliferation assay, and the data normalized to the biological activity of, the control (hGH solution at time zero). In other assays hGH and HA-hGH were incubated in phosphate buffer saline in at 4, 37 and 50 degree C.
Nb2 cell proliferation assay: The growth of these cells is dependent on hGH or other lactogenic hormones. In a typical experiment 104 cells/well are plated in 96-well plate in the presence of different concentration of hGH or HA-hGH in media such as DMEM containing 5-10% horse serum for 24-48 hrs in the incubator. After the incubation period, 1:10 volume of MTT (5 mg/ml in H2O) is added to each well and the plate is incubated for a further 6-16 hrs. The growing cells convert MTT to insoluble formazan. The formazan is solublized by acidic isopropanol, and the color produced is measured at 570 nm on microtiter plate reader. The extent of formazan formation reflects the level of cellular proliferation.
Increased Shelf-life of HA-hGH Fusion Proteins: Results
The fusion of Therapeutic proteins to albumin confers stability in aqueous or other solution. FIG. 1 depicts the extended shelf-life of an HA fusion protein in terms of the biological activity of HA-hGH remaining after storage in cell culture media for up to 5 weeks at 37° C. A solution of 200 μg/ml HA-hGH was prepared in tissue culture media containing 5% horse serum, and the solution incubated at 37° C. starting at time zero. At the indicated times, a sample was removed and tested for its biological activity in the Nb2 cell assay, at 2 ng/ml final concentration. As shown in FIG. 1, the biological activity of HA-hGH remains essentially intact (within experimental variation) after 5 weeks of incubation at 37° C. The recombinant hGH used as control for this experiment lost its biological activity in the first week of the experiment.
FIG. 2 shows the stability of HA-hGH after storage in cell culture media for up to 3 weeks at 4, 37, or 50° C. At time zero, a solution of HA-hGH was prepared in tissue culture media containing 5% horse serum, and incubated at 4, 37, and 50° C. At the indicated periods a sample was removed and assayed for its biological activity in the Nb2 cell proliferation assay, at 60 ng/ml final concentration. HA-hGH retains over 90% of its initial activity at all temperatures tested for at least 3 weeks after incubation while hGH loses its biological activity within the first week. This level of activity is further retained for at least 7 weeks at 37° C. and weeks at 50° C. These results indicate that HA-hGH is highly stable in aqueous solution even under temperature stress.
FIGS. 3A and 3B show the stable biological activity of HA-hGH compared to hGH in the Nb2 cell proliferation assay. Nb2 cells were grown in the presence of increasing concentrations of recombinant hGH or HA-hGH, added at time zero. The cells were incubated for 24 or 48 hours before measuring the extent of proliferation by the MTT method. The increased stability of HA-hGH in the assay results in essentially the same proliferative activity at 24 hours (FIG. 3A) as at 48 hours (FIG. 3B) while hGH shows a significant reduction in its proliferative activity after 48 hours of incubation (FIGS. 3A and 3B). Compared to hGH, the HA-hGH has lower biological potency after 1 day; the albumin fusion protein is about 5 fold less potent than hGH. However, after 2 days the HA-hGH shows essentially the same potency as hGH due to the short life of hGH in the assay. This increase in the stability of the hGH as an albumin fusion protein has a major unexpected impact on the biological activity of the protein. Although the potency of the albumin fusion proteins is slightly lower than the unfused counterparts in rapid bioassays, their biological stability results in much higher biological activity in the longer term in vitro assay or in vivo assays.
Example 2
Preparation of HA-fusion Proteins
FIG. 4 shows a map of a plasmid (pPPC0005) that can be used as the base vector for cloning the cDNAs of therapeutic partners to form HA-fusions. For example, digestion of this vector with the restriction enzymes Bsu361/Partial HindIII will allow for the insertion of a cDNA modified at the 5′ end to encode the last 5 amino acids of HA including the Bsu361 site and at the 3′ end to include a double stop codon and HindIII site. As another example, digestion of this vector with the restriction enzymes Bsu361; SphI allows for the insertion of a cDNA modified at the 5′ end to encode the last 5 amino acids of HA including the Bsu361 site and at the 3′ end to include a double stop codon, HindIII site and the ADH1 terminator sequence up to and including the SphI site.
This plasmid may easily be modified by one of skill in the art, for example, to modify, add or delete restriction sites so that one may more easily clone a Therapeutic protein, or fragment or variant of into the vector for the purpose of making an albumin fusion protein of the invention.
For example, for the purpose of making an albumin fusion protein where the Therapeutic moiety is placed N-terminal to the (mature) albumin protein, restriction sites were added at the 5′ end of the DNA encoding HA in pPPC0005 shown in FIG. 4).
Because it was desired to add unique XhoI and ClaI sites at the 5′ end of the DNA encoding the HA protein in pPPC0005, it was first necessary to remove those same sites from thee plasmid (located 3′ of the ADH1 terminator sequence. This was accomplished by cutting pPPC0005 with XhoI and ClaI, filling in the sticky ends with T4 DNA polymerase, and religating the blunt ends to create pPPC0006.
Engineering the Xho and Cla I restriction sites into the Fusion leader sequence just 5′ of the DNA encoding the HA protein in pPPC0006 was accomplished using two rounds of PCR. The first pair of oligonucleotides are those of SEQ ID NO:19 and SEQ ID NO:20. SEQ ID 19 contains four point mutations relative to the DNA sequence encoding the Fusion leaadr sequence and the beginning of the HA protein. These mutations are necessary to create the XhoI site in the fusion leader sequence and the Cla I site just at the beginning of the DNA encoding the HA protein. These four mutations are underlined in the sequence shown below. In pPPC0006 the nucleotides at these four positions from 5′ to 3′ are T, G, T, and G.

    
    
        5′-GCCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCGATTTAAAGATTTGGG-3′ (SEQ ID NO:19)
        5′-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3′ (SEQ ID NO:20).

A second round of PCR is then-performed with an upstream flanking primer, 5′-TACAAACTTAAGAGTCCAATTAGC-3′ (SEQ D NO:21) and a downstream flanking primer 5′-CACTTCTCTAGAGTGGTTTCATATGTCTT-3′ (SEQ ID NO:22). The resulting PCR product is then purified and then digested with AflI and XbaI and ligated into the same sites in pPPC0006 creating pScCHSA. The resulting plasmid will have an XhoI sites engineered into the fusion leader sequence. The presence of the XhoI site creates a single amino acid change in the end of fusion leader sequence from LDKR to LEKR. The D to E change will not be present in the final albumin fusion protein expression plasmid if one ligates into the XhoI and Cla I sites a fragment comprising the Therapeutic moiety which has a 5′ SalI sticky end (which is compatible with the XhoI end) and a 3° ClaI end. Ligation of the XhoI to the SalI restores the original amino acid sequence of the Fusion leader sequence. The Therapeutic protein moiety may be inserted after the Kex2 site (Kex2 claeves after the dibasic amino acid sequence KR at the end of the Fusion leader sequence) and before the ClaI site.

    
    


In addition, for the purpose of making an albumin fusion protein where the Therapeutic moiety is placed C-terminal to the (mature) albumin protein, four, eight-base-pair restriction sites were added at the 3′ end of the DNA encoding HA in pScCHSA. As an example, it was felt to be desirable to incorporate AscI, FseI, and PmeI restriction sites in between the Bsu361 and HindIII sites at the end of the DNA encoding the HA protein in pScCHSA. This was accomplished through the use of two complementary synthetic oligonucleotides (SEQ ID NO:19 and SEQ ID NO:20) which contain the desired restriction sites.

    
    
        5′-AAGCTGCCTTAGGCTTATAATAAGGCGCGCCGGCCGGCCGTTTAAACTAAGCTTAATTCT-3′ (SEQ ID NO:23) and
        5-AGAATTAAGCTTAGTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT-3′ (SEQ ID NO:24).

These oligonucleotides may be annealed and digested with Bsu361 and HindIII and ligated into the same sites located at the end of the DNA encoding the HA protein in pScCHSA creating pScNHSA, using techniques Known in the art.

    
    


Making Vectors Comprising Albumin Fusion Proteins where the Albumin Moiety is N-terminal to the Therapeutic Moiety.
The DNA encoding the Therapeutic moiety may be PCR amplified using primers that will add DNA encoding the last five amino acids of the HA (and containing the Bsu36I site) onto the 5′ end of the DNA encoding a Therapeutic protein and a STOP codon and appropriate cloning sites onto the 3′ end of the coding sequence. For instance, the forward primer used to amplify the DNA encoding a Therapeutic protein might have the sequence, 5′-aagctGCCTTAGGCTTA(N)15-3′ (SEQ ID NO:25) where the underlined sequence is a Bsu36I site, the upper case nucleotides encode the last four amino acids of the mature HA protein (ALGL), and (N)15 is identical to the first 15 nucleotides encoding the Therapetic protein of interest. Similarly, the reverse primer used to amplify the DNA encoding a Therapeutic protein might have the sequence, 




where the italicized nucleotides is a PmeI site, the double underlined nucleotides are a FseI site, the singly underlined text is a PmeI site, the boxed nucleotides are the reverse complement of two tandem stop codons, and (N)15 is identical to the reverse complement of the last 15 nucleotides encoding the Therapeutic protein of interest. Once the PCR product is amplified it may be cut with Bsu36I and one of (AscI, FseI, or PmeI) and ligated into pScNHSA.

Marking Vectors Comprising Albumin Fusion Proteins where the Albumin Moiety is N-terminal to the Therapeutic Moiety.
The DNA encoding the Therapeutic moiety may be PCR amplified using primers that will add DNA encoding the last three amino acids of the Fusion leader sequence (and containing a SalI site) onto the 5′ end of the DNA encoding a Therapeutic protein and the first few amino acids of the HA (and containing a ClaI site. For instance, the forward primer used to amplify the DNA encoding a Therapeutic protein might have the sequence, 5′-aggagcgtcGACAAAAGA(N)15-3′ (SEQ ID NO:27) where the underlined sequence is a Sal I site, the upper case nucleotides encode the last three amino acids of the Fusion leader sequence (DKR), and (N)15 is identical to the first 15 nucleotides encoding the Therapeic protein of interest Similarly, the reverse primer used to amplify the DNA encoding a Therapeutic protein might have the sequence, 5′-CTTTAAATCGATGAGCAACCTCACTCTTGTGTGCATC(N)15-3′ (SEQ ID NO:28) where the italicized nucleotides are a ClaI site, the underlined nucleotides are the reverse complement of the DNA encoding the first 9 amino acids of HA (DAHKSEV AH), and (N)15 is identical to the reverse complement of the last 15 nucleotides encoding the Therapeutic protein of interest. Once the PCR product is amplified it may be cut with SalI and ClaI and ligated into pScCHSA digested with XhoI and Cla I.
Expression of an Albumin Fusion Protein in Yeast.
The Not I fragment containing the DNA encoding either an N-terminal or C-terminal albumin fusion, protein generated from pScCHSA or pScNHSA may then be cloned in to the NotI site of pSAC35.
Expression of an Albumin Fusion Protein from Mammalian Cell Lines
The HSA gene has also been cloned into a the pC4 vector which is more suitable for mammalian culture systems creating plasmid pC4:HSA. More specifically, pC4HSA was generated by PCR amplifying the mature HSA gene with a 5′ primer (SEQ ID NO:30) that anneals to the 5′ end of DNA encoding the mature form of the HSA protein (e.g., DNA in plasmid pScCHSA), incorporates BamHI (Shown in italics below) and HindIII (shown singly underlined below) cloning sites, attaches a kozak sequence (shown double underlined below) and DNA encoding the natural HSA signal peptide (MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:29) (shown in bold below), and a 3′ primer (SEQ ID NO:3 1) that anneals to the 3′ end of DNA encoding the mature form of the HSA protein and incorporates an Asp718 restriction site (shown in bold below). The DNA encoding the natural human serum albumin leader sequence in SEQ ID NO:30 also contains a modification that introduces a XhoI site that is boxed below. 




This PCR product (1.85 kb) is then purified and digested with BamHI and Asp718 and cloned into the same sites in pC4 (ATCC Accession No. 209646) to produce pC4:HSA
Making Vectors Comprising Albumin Fusion Proteins where the Albumin Moirty is C-terminal to the Therapeutic Moiety Using the pC4:HSA Vector
Using pC4:HSA, albumin fusion proteins in which the Therapeutic protein moiety is N terminal to the albumin sequence, one can clone DNA encoding a Therapeutic protein that has its own signal sequence between the Barn HI (or HindIII) and ClaI sites. When cloning into either the BamHI or HindIII site remember to include Kozak sequence (CCGCCACCATG) prior to translational start codon of DNA encoding the Therapeutic Protein to be subcloned. If the Therapeutic does not have a signal sequence, the DNA encoding that Therapeutic protein may be cloned in between the XhoI and ClaI sites. When using the XhoI site, the following 5′ (SEQ ID NO:32) and 3′ (SEQ ID NO:33) PCR primers may be used:

    
    
        5′-CCGCCGCTCGAGGGGTGTGTTTTCGTCGA(N)18-3′ (SEQ ID NO: 32)
        5′-AGTCCCATCGATGAGCAACCTCACTCTTGTGCATC(N)18-3′ (SEQ ID NO:33)
    
    


In SEQ ID NO:32, the underlined sequence is an XhoI site; and the XhoI site and the DNA following the XhoI site encode for the last seven amino acids of the leader sequence of natural human serum albumin. In SEQ ID NO:33, the underlined sequence is a ClaI site; and the ClaI site and the DNA following it encode are the reverse complement of the DNA encoding the first 10 amino acids of themature HSA protein (SEQ ID NO: 18) In SEQ ID NO:32 “(N)18” is DNA identical to the first 18 nucleotides encoding the Therapeutic protein of interest.). In SEQ ID NO:33 “(N)18” is the reverse complement of DNA encoding the last 18 nucleotides encoding the Therapeutic protein of interest. Using these two primers, one may PCR amplify the Therapeutic protein of interest, purify the PCR product, digest it with XhoI and ClaI restriction enzymes and then and clone it into the with XhoI and ClaI sites in the pC4:HSA vector.
Making Vectors Comprising Albumin Fusion Proteins where the Albumin Moiety is N-terminal to the Therapeutic Moiety Using the pC4:HSA Vector
Using pC4:HSA, albumin fusion proteins in which the Therapeutic protein moiety is N terminal to the albumin sequence, one can clone DNA encoding a Therapeutic protein between the Bsu36I and AscI restriction sites. When cloning into the Bsu36I and AscI, the same primer design used to clone in the yeast vector system (SEQ ID NO:25 and 26) may be employed.
The pC4 vector is especially suitable for expression of albumin fusion proteins from CHO cells. For expression, in other mammalian cell types, e.g., NSO cells, it may be useful to subclone the HindIII-EcoRI fragment containing the DNA encoding an albumin fusion protein (from a pC4 vector in which the DNA encoding the Therapeutic protein has already been cloned in frame with the DNA encoding (the mature form of) human serum albumin) into another expression vector (such as any of the mammalian expression vectors described herein).
Example 3
Preparation of HA-cytokine or HA-growth Factor Fusion Proteins (Such as EPO, GMCSF, GCSF)
The cDNA for the cytokine or growth factor of interest, such as EPO, can be isolated by a variety of means including from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in U.S. Pat. Nos. 4,703,008, 4,810,643 and 5,908,763. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. EPO (or other cytokine) cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines, a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 4
Preparation of HA-IFN Fusion Proteins (Such as IFNα)
The cDNA for the interferon of interest such as IFNα can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for interferons, such as IFNα are known and available, for instance, in U.S. Pat. Nos. 5,326,859 and 4,588,585, in EP 32 134, as well as in public databases such as GenBank. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used to clone the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus of the HA sequence with or without the use of a spacer sequence. The IFNα (or other interferon) cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast (see FIG. 8). The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Maximum Protein Recovery from Vials
The albumin fusion proteins of the invention have a high degree of stability even when they are packaged at low concentrations. In addition, in spite of the low, protein concentration good fusion-protein recovery is observed even when the aqueous solution includes no other protein added to minimize binding to the vial walls. FIG. 5 compares the recovery of vial-stored HA-IFN solutions with a stock solution 6 or 30 μg/ml HA-IFN solutions were placed in vials and stored at 4° C. After 43 or 72 hrs a volume originally equivalent to 10 ng of sample was removed and measured in an IFN sandwich ELISA. The estimated values were compared to that of a high concentration stock solution. As shown, there is essentially no loss of the sample in these vials, indicating that addition of exogenous material such as albumin is not necessary to prevent sample loss to the wall of the vials.
In vivo Stability and Bioavailability of HA-α-IFN Fusions
To determine the in vivo stability and bioavailability of a HA-α-IFN fusion molecule, the purified fusion molecule (from yeast) was administered to monkeys at the dosages and time points described in FIGS. 6 and 7. Pharmaceutical compositions formulated from HA-α-IFN fusions may account for the extended serum half-life and bioavailability exemplified in FIGS. 6 and 7. Accordingly, pharmaceutical compositions may be formulated to contain lower dosages of alpha-interferon activity compared to the native alpha-interferon molecule.
Pharmaceutical compositions containing HA-α-IFN fusions may be used to treat or prevent disease in patients with any disease or disease state that can be modulated by the administration of α-IFN. Such diseases include, but are not limited to, hairy cell leukemia, Kaposi's, sarcoma, genital and anal warts, chronic hepatitis B, chronic non-A, non-B hepatitis, in particular hepatitis C, hepatitis D, chronic myelogenous leukemia, renal cell carcinoma, bladder carcinoma, ovarian and cervical carcinoma, skin cancers, recurrent respirator papillomatosis, non-Hodgkin's and cutaneous T-cell lymphomas, melanoma, multiple myeloma, AIDS, multiple sclerosis, gliobastoma, etc. (see Interferon Alpha, In: AHFS Drug Information, 1997.
Accordingly, the invention includes pharmaceutical compositions containing a HA-α-IFN fusion protein, polypeptide or peptide formulated with the proper dosage for human administration. The invention also includes methods of treating patients in need of such treatment comprising at least the step of administering a pharmaceutical composition containing at (east one HA-α-IFN fusion protein, polypeptide or peptide.
Bifunctional HA-_-IFN Fusions
The HA-α-IFN expression vector of FIG. 8 is modified to include an insertion for the expression of bifunctional HA-α-IFN fusion proteins. For instance, the cDNA for a second protein of interest may be inserted in frame downstream of the “rHA-IFN” sequence after the double stop codon has been removed or shifted downstream of the coding sequence.
In one version of a bifunctional HA-α-IFN fusion protein, an antibody or fragment against B-lymphocyte stimulator protein (GenBank Acc 4455139) or polypeptide may be fused to one end of the HA component of the fusion molecule. This bifunctional protein is useful for modulating any immune response generated by the α-IFN component of the fusion.
Example 5
Preparation of HA-hormone Fusion Protein (Such as Insulin, LH, FSH)
The cDNA for the hormone of interest such as insulin can be isolated by a variety of means including, but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in public databases such as GenBank. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be-used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The hormone cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 6
Preparation of HA-soluble Receptor or HA-binding, Protein Fusion Protein Such as HA-TNF Receptor
The cDNA for the soluble receptor or binding protein of interest such as TNF receptor can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for all of these proteins are known and available, for instance, in GenBank. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The receptor cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 7
Preparation of HA-growth Factors Such as HA-IGF-1 Fusion Protein
The cDNA for the growth factor of interest such as IGF-1 can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods (see GenBank Acc. No.NP—00609). The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The growth factor cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 8
Preparation of HA-single Chain Antibody Fusion Proteins
Single chain antibodies are produced by several methods including but not limited to: selection from phase libraries, cloning of the variable region of a specific antibody by cloning the cDNA of the antibody and using the flanking constant regions as the primer to clone the variable region, or by synthesizing an oligonucleotide corresponding to the variable region of any specific antibody. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The cell cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast.
In fusion molecules of the invention, the VH and VL can be linked by one of the following means or a combination thereof: a peptide linker between the C-terminus of the VH and the N-terminus of the VL; a Kex2p protease cleavage site between the VH and VL such that the two are cleaved apart upon secretion and then self associate; and cystine residues positioned such that the VH and VL can form a disulphide bond between them to link them together (see FIG. 14). An alternative option would be to place the VH at the N-terminus of HA or an HA domain fragment and the VL at the C-terminus of the HA or HA domain fragment.
The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its activity. For expression in mammalian cell lines a similar procedure is adopted-except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines. The antibody produced in this manner can be purified from media and tested for its binding to its antigen using standard immunochemical methods.
Example 9
Preparation of HA-cell Adhesion Molecule Fusion Proteins
The cDNA for the cell adhesion molecule of interest can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The nucleotide sequences for the known cell adhesion molecules are known and available, for instance, in GenBank. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The cell adhesion molecule cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 10
Preparation of Inhibitory Factors and Peptides as HA Fusion Proteins (Such as HA-antiviral, HA-antibiotic, HA-enzyme Inhibitor and HA-anti-allergic Proteins)
The cDNA for the peptide of interest such as an antibiotic peptide can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers all using standard methods. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for HA. This can be at the N or C-terminus with or without the use of a spacer sequence. The peptide cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 11
Preparation of Targeted HA Fusion Proteins
The cDNA for the protein of interest can be isolated from cDNA library or can be made synthetically using several overlapping oligonucleotides using standard molecular biology methods. The appropriate nucleotides can be engineered in the cDNA to form convenient restriction sites and also allow the attachment of the protein cDNA to albumin cDNA similar to the method described for hGH. Also a targeting protein or peptide cDNA such as single chain antibody or peptides, such as nuclear localization signals, that can direct proteins inside the cells can be fused to the other end of albumin. The protein of interest and the targeting peptide is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA which allows the fusion with albumin cDNA. In this manner both N- and C-terminal end of albumin are fused to other proteins. The fused cDNA is then excised from pPPC0005 and is inserted into a plasmid such as pSAC35 to allow the expression of the albumin fusion protein in yeast. All the above procedures can be performed using standard methods in molecular biology. The albumin fusion protein secreted from yeast can be collected and purified from the media and tested for its biological activity and its targeting-activity using appropriate biochemical and biological tests.
Example 12
Preparation of HA-enzymes Fusions
The cDNA for the enzyme of interest can be isolated by a variety of means including but not exclusively, from cDNA libraries, by RT-PCR and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods. The cDNA can be tailored at the 5′ and 3′ ends to generate restriction sites, such that oligonucleotide linkers can be used, for cloning of the cDNA into a vector containing the cDNA for IL, This can be at the N or C-terminus with or without the use of a spacer sequence the enzyme cDNA is cloned into a vector such as pPPC0005 (FIG. 4), pScCHSA, pScNHSA, or pC4:HSA from which the complete expression cassette is then excised and inserted into the plasmid pSAC35 to allow the expression of the albumin fusion protein in yeast. The albumin fusion protein secreted from the yeast can then be collected and purified from the media and tested for its biological activity. For expression in mammalian cell lines a similar procedure is adopted except that the expression cassette used employs a mammalian promoter, leader sequence and terminator (See Example 2). This expression cassette is then excised and inserted into a plasmid suitable for the transfection of mammalian cell lines.
Example 13
Bacterial Expression of an Albumin Fusion Protein
A polynucleotide encoding an albumin fusion protein of the present invention comprising a bacterial signal sequence is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, to synthesize insertion fragments. The primers used to amplify the polynucleotide encoding insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to, the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thioalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl or preferably in 8 M urea and concentrations greater than 0.14 M 2-mercaptoethanol by stirring for 34 hours at 4° C. (see, e.g., Burton et al., Eur. J. Biochem. 179:379-387 (1989)). The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid.(“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. Exemplary conditions are as follows: renature using a linear 6M-1M urea gradient in 500 m NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4° C. or frozen at −80° C.
In addition to the above expression vector, the present invention further includes an expression vector, called pHE4a (ATCC Accession Number 209645, deposited on Feb. 25, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide encoding an albumin fusion protein of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter and operator sequences are made synthetically.
DNA can be inserted into the pHE4a by restricting the vector with NdeI and. XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to PCR protocols described herein or otherwise known in the art, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector may be substituted in the above protocol to express protein in a bacterial system.
Example 14
Expression of an Albumin Fusion Protein in Mammalian Cells
The albumin fusion proteins of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, but are not limited to, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the albumin fusion protein can be expressed in stable cell lines containing the polynucleotide encoding the albumin fusion protein integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
The transfected polynucleotide encoding the fusion protein can also be amplified to express large amounts of the encoded fusion protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin et al., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page et al., Biotechnology 64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10: 169-175 (1992). Using, these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
Derivatives of the plasmid pSV2dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
A polynucleotide encoding an albumin fusion protein of the present invention is generated using techniques known in the art and this polynucleotide is amplified using PCR technology known in the art. If a naturally occurring signal sequence is used, to produce the fusion protein of the present invention, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, egg., International Publication No. WO 96/34891.)
The amplified fragment encoding the fusion protein of the invention is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment encoding the albumin fusion protein of the invention is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five μg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired fusion protein is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 15
Multifusion Fusions
The albumin fusion proteins (e.g., containing a Therapeutic protein (or fragment or variant thereof) fused to albumin (or a fragment or variant thereof)) may additionally be fused to other proteins to generate “multifusion proteins”. These multifusion proteins can be used for a variety of applications. For example, fusion of the albumin fusion proteins of the invention to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See e.g., EP A 394,827; Traunecker et al., Nature 331:84-86 (1988)). Nuclear localization signals fused to the polypeptide of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of an albumin fusion protein. Furthermore, the fusion of additional protein sequences to the albumin fusion proteins of the invention may further increase the solubility and/or stability of the fusion protein. The fusion proteins described above can be made using or routinely modifying techniques known in the art and/or by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian or yeast expression vector.
For example, if pC4 (ATCC Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide encoding an albumin fusion protein of the present invention (generateed and isolated using techniques known in the art), is ligated into this BamHI site. Note that the polynucleotide encoding the fusion protein of the invention is cloned without a stop codon, otherwise a Fc containing fusion protein will not be produced.
If the naturally occurring signal sequence is used to produce the albumin fusion protein of the present invention, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)
Human IgG Fc region:

    
    
        GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCFTCGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO: 36)
    
    


Example 16
Production of an Antibody from an Albumin: Fusion Protein
a) Hybridoma Technology
Antibodies that bind the albumin fusion proteins of the present invention and portions of the albumin fusion proteins of the present invention (e.g., the Therapeutic protein portion or albumin portion of the fusion protein) can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, a preparation of an albumin fusion protein of the invention or a portion of an albumin fusion protein of the invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, are prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention.
Alternatively, additional antibodies capable of binding to an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the an albumin fusion protein of the invention (or portion of an albumin fusion protein of the invention)—specific antibody can be blocked by the fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Such antibodies comprise anti-idiotypic antibodies to the fusion protein of the invention (or portion of an albumin fusion protein of the invention)—specific antibody and are used to immunize an animal to induce formation of further fusion protein of the invention (or portion of an albumin fusion protein of the invention)—specific antibodies.
For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).
b) Isolation of Antibody Fragments Directed Against an Albumin Fusion Protein of the Invention, or a Portion of an Albumin Fusion Protein of the Invention from A Library of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).
Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-ANP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see International Publication No. WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in International Publication No. WO 92/01047.
M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phase in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and crown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1996), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS. 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with miciotitre plates coated with either 10 pg/ml of an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further a characterized by PCR fingerprinting (see, e.g., International Publication No. WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
Example 17
Method of Treatment Using Gene Therapy-ex vivo
One method of gene therapy transplants fibroblasts, which are capable of expressing an albumin fusion protein of the present invention, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
Polynucleotides encoding an albumin fusion protein of the invention can be generated using techniques known in the art amplified using PCR primers which correspond to the 5′ and 3′ end sequences and optionally having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.
The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Mviedium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether the albumin fusion protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
Example 18
Method of Treatment Using Gene Therapy—in vivo
Another aspect of the present invention is using in vivo gene therapy methods to treat isorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences encoding an albumin fusion protein of the invention into an animal. Polynucleotides encoding albumin fusion proteins of the present invention may be operatively linked to (i.e., associated with) a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).
The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, polynucleotides encoding albumin fusion proteins of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct can be delivered to the interstitial space of tissues within an animal, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin isclosed with stainless steel clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for fusion protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 19
Transgenic Animals
The albumin fusion proteins of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express fusion proteins of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the polynucleotides encoding the albumin fusion proteins of the invention into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotecbnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones containing polynucleotides encoding albumin fusion proteins of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence. (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
The present invention provides for transgenic animals that carry the polynucleotides encoding the albumin fusion proteins of the invention in all their cells, as well as animals which carry these polynucleotides in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide encoding the fusion protein of the invention be integrated into the chromosomal site of the endogenous gene corresponding to the Therapeutic protein portion or albumin portion of the fusion protein of the invention, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the polynucleotide encoding the fsuion protien of the invention has taken place. The level of mRNA expression of the polynucleotide encoding the fusion protein of the invention in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of fusion protein-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the fusion protein.
Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene (i.e., polynucleotide encoding an albumin fusion protein of the invention) on a distinct background that is appropriate for an experimental model of interest.
Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of fusion proteins of the invention and the Therapeutic protein and/or albumin component of the fusion protein of the invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 20
Assays Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation
Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvirorment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing tolerance and death among B cell populations.
One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.
In Vitro Assay—Albumin fusion proteins of the invention (including fusion proteins, containing fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin) can be assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of an albumin fusion protein of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).
Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 105 B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10−5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10−5 dilution of SAC) in a total volume of 150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.
In vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of an albumin fusion protein of the invention (including fusion proteins containing fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin). Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with the albumin fusion protein of the invention identify the results of the activity of the fusion protein on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.
Flow cytometric analyses of the spleens from mice treated with the albumin fusion protein is used to indicate whether the albumin fusion protein specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.
Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and fusion protein treated mice.
The studies described in this example tested activity of fusion proteins of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins and polynucleotides of the invention (e.g., gene therapy).
Example 21
T Cell Proliferation Assay
A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of 3H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of an albumin fusion protein of the invention (including fusion proteins containing fragments or variants of Therapeutic proteins and/or albumin or fragments or variants of albumin) (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatant is removed and stored −20 degrees C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of 3H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells are harvested and incorporation of 3H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative control for the effects of fusion proteins of the invention.
The studies described in this example tested activity of fusion proteins of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins or polynucleotides of the invention (e.g., gene therapy).
Example 22
Effect of Fusion Proteins of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-derived Human Dendritic Cells
Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.
FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of an albumin fusion protein of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Thl helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (106/ml) are treated with increasing concentrations of an albumin fusion protein of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.
Effect on the expression of MHC Class II, costinulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increased expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.
FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of an albumin fusion protein of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide; and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Albumin fusion proteins of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.
Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated processes (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the fusion protein to be tested. Cells are suspended at a concentration of 2×106/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.
Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×105 cells/ml with increasing concentrations of an albumin fusion protein of the invention and under the same conditions, but in the absence of the fusion protein. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in the presence of the fusion protein. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.
Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×105 cell/well. Increasing concentrations of an albumin fusion protein of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37° C. for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H2O2 produced by the macrophages, a standard curve of a H2O2 solution of known molarity is performed for each experiment.
The studies described in this example tested activity of fusion proteins of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins or polynucleotides of the invention (e.g., gene therapy).
Example 23
Biological Effects of Fusion Proteins of the Invention
Astrocyte and Neuronal Assays
Albumin fusion proteins of the invention can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate an albumin fusion protein of the invention's activity on these cells.
Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety) However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of an albumin fusion protein of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.
Fibroblast and Endothelial Cell Assays
Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test fusion protein of the invention proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE2 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or fusion protein of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without an albumin fusion protein of the invention and/or IL-1α for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).
Human lung fibroblasts are cultured with FGF-2 or an albumin fusion protein of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with the fusion protein of the invention.
Cell Proliferation Based on [3H]thymidine Incorpration
The following [3H]Thymidine incorporation assay can be used to measure the effect of a Therapeutic proteins, e.g., growth factor proteins, on the proliferation of cells such as fibroblast cells, epithelial cells or immature muscle cells. Sub-confluent cultures are arrested in G1 phase by an 18 h incubation in serum-free medium. Therapeutic proteins are then added for 24 h and during the last 4 h, the cultures are labeled with [3H]thymidine, at a final concentration of 0.33 μM (25 Ci/mmol, Amersham, Arlington Heights, Ill.). The incorporated [3H]thymidine is precipitated with ice-cold 10% trichloroacetic acid for 24 h. Subsequently, the cells are rinsed sequentially with ice-cold 10% trichloroacetic acid and then with ice-cold water. Following lysis in 0.5 M NaOH, the lysates and PBS rinses (500 ml) are pooled, and the amount of radioactivity is measured.
Parkinson Models.
The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopamiinergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl4 phenyl 1,2,3,6-tetrahydropyridine (MFTP). In the CNS, MFTP is taken-up by astrocytes and cataholized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released. Subsequently, MPP+ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP+ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex 1), thereby interfering with electron transport and eventually generating oxygen radicals.
It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).
Based on the data with FGF-2, an albumin fusion protein of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of an albumin fusion protein of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.
Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if a Therapeutic protein acts to prolong the survival of dopaminergic neurons, it would suggest that the fusion protein may be involved in Parkinson's Disease.
The studies described in this example tested activity of albumin fusion proteins of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins and polynucleotides of the invention (e.g., gene therapy).
Example 24
The Effect of Albumin Fusion Proteins of the Invention on the Growth of Vascular Endothelial Cells
On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. An albumin fusion protein of the invention, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that the fusion protein may proliferate vascular endothelial cells, while a decrease in the number of HUVEC cells indicates that the fusion protein inhibits vascular endothelial cells.
The studies described in this example tested activity of an albumin fusion protein of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of a fusion protiem and polynucleotides of the invention.
Example 25
Rat Corneal Wound Healing Model
This animal model shows the effect of an albumin fusion protein of the invention on neovascularization. The experimental protocol includes:

    
    
        Making a 1-1.5 mm long incision from the center of cornea into the stromal layer.
        Inserting a spatula below the lip of the incision facing the outer corner of the eye.
        Making a pocket (its base is 1-1.5 mm form the edge of the eye).
        Positioning a pellet, containing 50 ng-5 ug of an albumin fusion protein of the invention, within the pocket.
        Treatment with an an albumin fusion protein of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).
    
    


The studies described in this example test the activity of an albumin fusion protein of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins and polynucleotides of the invention (e.g., gene therapy).
Example 26
Diabetic Mouse and Glucocorticoid-impaired Wound Healing Models
Diabetic db+/db+ Mouse Model.
To demonstrate that an albumin fusion protein of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
An albumin fusion protein of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an albumin fusion protein of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.
Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.
Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant
Steroid Impaired Rat Model
The inhibition of wound healingo by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Anti-inflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Acadermic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
To demonstrate that an albumin fusion protein of the invention can accelerate the healing process, the effects of multiple topical applications of the fusion protein on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.
Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
The wounding protocol is followed according to that described above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
The fusion protein of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an albumin fusion protein of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.
Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
The studies described in this example tested activity of an albumin fusion protein of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins and polynucleotides of the invention (e.g., gene therapy).
Example 27
Lymphedema Animal Model
The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of an albumin fusion protein of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.
Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately  ̃350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.
Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and heinostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated or suture ligated.
Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.
Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of  ̃0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.
To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect of plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.
Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people and those 2 readings are averaged. Readings are taken from both control and edematous limbs.
Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), and both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software(Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.
Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior tosurgery and then at conclusion for total protein and Ca2+ comparison.
Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.
Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80EC. until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.
The studies described in this example tested activity, of fusion proteins of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion protein and polynucleotides of the invention (e.g., gene therapy).
Example 28
Suppression of TNF Alpha-induced Adhesion Molecule Expression by an Albumin Fusion Protein of the Invention
The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.
The potential of an albumin fusion protein of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.
To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO2. HUVECs are seeded in 96-well plates at concentrations of 1×104 cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.
Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.
Fixative is then removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.
Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer 1:5,000 (100)>10−0.5>10−1>10−1.50.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
The studies described in this example tested activity of fusion proteins of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of fusion proteins and polynucleotides of the invention (e.g., gene therapy).
Example 29
Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class 1, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.
The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Damell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 37)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.













GAS











JAKs

(elements)













Ligand
tyk2
Jak1
Jak2
Jak3
STATS
or ISRE





IFN family








IFN-a/B
+
+
−
−
1,2,3
ISRE


IFN-g

+
+
−
1
GAS








(IRF1 >








Lys6 >








IFP)


I1-10
+
?
?
−
1, 3


gp130 family


IL-6 (Pleiotropic)
+
+
+
?
1, 3
GAS








Lys6 >








IFP)


II-11(Pleiotropic)
?
+
?
?
1, 3


OnM(Pleiotropic)
?
+
+
?
1, 3


LIF(Pleiotropic)
?
+
+
?
1, 3


CNTF(Pleiotropic)
−/+
+
+
?
1 ,3


G-CSF(Pleiotropic)
?
+
?
?
1, 3


IL-12(Pleiotropic)
+
−
+
+
1, 3


g-C family


IL-2 (lymphocytes)
−
+
−
+
1, 3, 5
GAS


IL-4 (lymph/myeloid)
−
+
−
+
6
GAS








(IRF1 =








IFP >>








(IgH)


IL-7 (lymphocytes)
−
+
−
+
5
GAS


IL-9 (lymphocytes)
−
+
−
+
5
GAS


IL-13 (lymphocyte)
−
+
?
?
6
GAS


IL-15
?
+
?
+
5
GAS


gp140 family


IL-3 (myeloid)
−
−
+
−
5
GAS








(IRF1 >








IFP >>








Ly6)


IL-5 (myeloid)
−
−
+
−
5
GAS


GM-CSF (myeloid)
−
−
+
−
5
GAS


Growth


hormone


family


GH
?
−
+
−
5


PRL
?
+/−
+
−
1, 3, 5


EPO
?
−
+
−
5
GAS








(B-CAS >








(IRF1 =








IFP >








Ly6)


Receptor


Tyrosine


Kinases


EGF
?
+
+
−
1, 3
GAS








(IRF1)


PDGF
?
+
+
−
1, 3


CSF-1
?
+
+
−
1, 3
GAS








(not IRF1)









To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 32-33, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is:

    
    
        5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′ (SEQ ID NO: 38)
    
    


The downstream primer is complementary to the SV40 promoter and is flanked with a HindIII site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 39)
PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/HindIII and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:

    
    
        5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′ (SEQ ID NO: 40)
    
    


With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), lticiferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Ciontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 32-33.
Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing EGR and NF-KB promoter sequences are described in Examples 34 and 35. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 30
Assay for SEAP Activity
As a reporter molecule for the assays described in examples disclosed herein, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5×Dilution Buffer and dispense 15 ul of 2.5×dilution buffer into Optiplates containing 35 ul of a solution containing an albumin fusion protein of the invention. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.










Reaction Buffer Formulation:









# of plates
Rzn buffer diluent (ml)
CSPD (ml)












10
 60
3


11
 65
3.25


12
 70
3.5


13
 75
3.75


14
 80
4


15
 85
4.25


16
 90
4.5


17
 95
4.75


18
100
5


19
105
5.25


20
110
5.5


21
115
5.75


22
120
6


23
125
6.25


24
130
6.5


25
135
6.75


26
140
7


27
145
7.25


28
150
7.5


29
155
7.75


30
160
8


31
165
8.25


32
170
8.5


33
175
8.75


34
180
9


35
185
9.25


36
190
9.5


37
195
9.75


38
200
10


39
205
10.25


40
210
10.5


41
215
10.75


42
220
11


43
225
11.25


44
230
11.5


45
235
11.75


46
240
12


47
245
12.25


48
250
12.5


49
255
12.75


50
260
13









Example 31
Assay Identifying Neuronal Activity
When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The prometer of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, the ability of fusion proteins of the invention to activate cells can be assessed.
Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by an albumin fusion protein of the present invention can be assessed.
The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al., Oricogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:

    
    
        5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO: 41)
        5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′ (SEQ ID NO: 42)
    
    


Using the GAS:SEAP/Neo vector produced in Example 29, EGR1 amplified product can then be-inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.
To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC12 using techniques known in the art. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 u/mL G418 for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×105 cells/ml.
Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1×105 cells/well). Add a series of different concentrations of an albumin fusion protein of the inventon, 37 degree C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay may be routinely performed using techniques known in the art and/or as described in Example 30.
Example 32
Assay for T-cell Activity
The following protocol is used to assess T-cell activity by identifying factors, and determining whether an albumin fusion protein of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 29. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.
During the incubation period, count cell concentration, spin down the required number of cells (107 per transfection), and resuspend in OPTI-MEM to a final concentration of 107 cells/ml. Then add 1 ml of 1×107 cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with varying concentrations of one or more fusion proteins of the present invention.
On the day of treatment with the fusion protein, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of fusion proteins and the number of different concentrations of fusion proteins being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.
The well dishes containing Jurkat cells treated with the fusion protein are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at −20 degree C. until SEAP assays are performed according to Example 30. The plates containing the remaining treated cells are placed at 4 degree C. and serve as a source of material for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.
The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.
Example 33
Assay for T-cell Activity
NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF-KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the fusion protein. Activators or inhibitors of NF-KB would be useful in treating, preventing, and/or diagnosing diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.
To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO: 43), 18 bp of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site:

    
    
        5′:GCGGCCTCGAGGGGATTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′ (SEQ ID NO: 44)
    
    


The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a HindIII site:

    
    
        5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 39)
    
    


PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and HindIII and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:

    
    
        5′:CTCGAGGGGACTTTCCCGGGGACTTTCGGGGGACTTTCCGGGATTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTATTTATGCAGAGGCCGAGGCCTCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′ (SEQ ID NO: 45)
    
    


Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using, XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.
Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 32. Similarly, the method for assaying fusion proteins with these stable Jurkat T-cells is also described in Example 32. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.
Example 33
Assay Identifying Myeloid Activity
The following protocol is used to assess myeloid activity of an albumin fusion protein of the present invention by determining whether the fusion protein proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 29. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 29, a DEAE-Dextran method (Kharbanda et al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×107 U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.
Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH-7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 MM KCl, 375 uM Na2HPO4.7H2O, 1 mM MgCl2, and 675 uM CaCl2. Incubate at 37 degrees C. for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degree C. for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages.
These cells are tested by harvesting 1×108 cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×105 cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×105 cells/well).
Add different concentrations of the fusion protein. Incubate at 37 degee C. for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to methods known in the art and/or the protocol described in Example 30.
Example 34
Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify fusion proteins which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.
For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO2 incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO2 incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×106 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×106 cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 ul, followed by an aspiration step to 100 ul final volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The fusion protein of the invention is added to the well, and a change in fluorescence is detected.
To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event caused by an albumin fusion protein of the present invention or a molecule induced by an albumin fusion protein of the present invention, which has resulted in an increase in the intracellular Ca++ concentration.
Example 35
Assay Identifying Tyrosine Kinase Activity
The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein. Tyrosine Kinase (RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether an albumin fusion protein of the present invention or a molecule induced by a fusion proetin of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or a different concentrations of an albumin fusion protein of the invention, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C. at 16,000×g.
Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.
Generally, the tyrosine kinase activity of an albumin fusion protein of the invention is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of 5 uM Biotinylated Peptide, then 10 ul ATP/Mg2+ (5 mM ATP/50 mM MgCl2), then 10 ul of 5×Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl2, 5 mM MnCl2, 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate(1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30 degree C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C. for 20 min. This allows the streptavidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37 degree C. for one hour. Wash the well as above.
Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 36
Assay Identifying Phosphorylation Activity
As a potential alternative and/or complement to the assay of protein tyrosine kinase activity described in Example 35, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), Mek kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.
Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degree C. until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or varying concentrations of the fusion protein of the invention for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by the fusion protein of the present invention or a molecule induced by an albumin fusion protein of the present invention.
Example 37
Assay for the Stimulation of Bone Marrow CD34+ Cell Proliferation
This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of fusion proteins of the inventon to stimulate proliferation of CD34+ cells.
It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of fusion proteins of the invention on hematopoiefic activity of a wide range of progenitor cells, the assay contains a given fusion protein of the invention in the presence or absence of hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a “survival” factor. However, combined with any factor exhibiting simulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested fusion protein has a stimulatory effect on hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect of a given fusion protein might not be detected. Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+IL+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.
Briefly, CD34+ cells are isolated using methods known in the art. The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md. Cat# 160-204-101). After several gentle centrifugation steps at 200×g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5×105 cells/ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with an albumin fusion protein of the invention in this assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat# 255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat# 203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, varying concentrations of an albumin fusion protein of the invention, and 20 μl of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 μl. The plates are then placed in a 37° C./5% CO2 incubator for five days.
Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H] Thymidine is added in a 10 μl volume to each well to determine the proliferation rate. The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OmniFilter assemblies consisting of one OmniFilter plate and one OmniFilter Tray. 60 μl Microscint is added to each well and the plate sealed with TopSeal-A press-on sealing film A bar code 15 sticker is affixed to the first plate for counting. The sealed plates are then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.
The studies described in this example test the activity of a given fusion protein to stimulate bone marrow CD34+ cell proliferation. One skilled in the art could easily modify the exemplified studies to test the activity of fusion porteins and polynucleotides of the invention (e.g., gene therapy) as well as agonists and antagonists thereof. The ability of an albumin fusion protein of the invention to stimulate the proliferation of bone marrow CD34+cells indicates that the albumin fusion protein and/or polynucleotides corresponding to the fusion protein are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein.
Example 38
Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
The objective of the Extracellula Matrix Enhanced Cell Response (EMECR) assay is to evaluate the ability of fusion proteins of the invention to act on hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.
Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fn is mediated by the α5.β1 and α4.β1 integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM environment and are responsible for stimulating stem cell self-renewal havea not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications
Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fn fragment at a coating concentration of 0.2 μg/cm2. Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Albumin fusion proteins of the invention are tested with appropriate negative controls in the presence and absence of SCF(5.0 ng/ml), where volume of the administed composition containing the albumin fusion protein of the invention represents 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment (5% CO2, 7% O2, and 88% N2) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thymidine incorporation into cellular DNA. Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.
One skilled in the art could easily modify the exemplified studies to test the activity of albumin fusion proteins and polynucleotides of the invention (e.g., gene therapy).
If a particular fusion protein of the present invention is found to be a stimulator of hematopoietic progenitors, the fusion protein and polynucleotides corresponding to the fusion protein may be useful for example, in the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein. The fusion protein may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
Additionally, the albumin fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.
Moreover, fusion proteins of the invention and polynucleotides encoding albumin fusion proteins of the invention may also be useful for the treatment and diagnosis of hematopoietic related disorders such as, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
Example 39
Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation
An albumin fusion protein of the invention is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the fusion protein on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity.
Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2%FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5%FBS. After incubation at 37° C. for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37° C. until day 2.
On day 2, serial dilutions and templates of an albumin fusion protein of the invention are designed such that they always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add 1⁄3 vol media containing controls or an albumin fusion protein of the invention and incubate at 37 degrees C./5% CO2 until day 5.
Transfer 60 μl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4 degrees C. until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10 μl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CytoFluor. This yields the growth stimulation/inhibition data.
On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.
On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 μA/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.
Plates are washed with wash buffer and blotted on paper towels, Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover the plate and incubate 1 h at RT. Plates are again washed with wash buffer and blotted on paper towels.
Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.
A positive result in this assay suggests AoSMC cell proliferation and that the albumin fusion protein may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of the fusion protein and polynucleotides encoding the albumin fusion protein. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, fusion proteins may be used in wound healing and dermal regeneration, as well as the promotion of vasculogenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, fusion proteins showing antagonistic activity in this assay may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular agent (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, albumin fusion proteins that act as antagonists in this assay may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.
Example 40
Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells
The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HLVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing (containing an albumin fusion protein of the invention) and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are then incubated at 37° C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min. Fixative is removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000 dilution, referred to herein as the working dilution) are added to each well and incubated at 37° C. for 30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (100)>10−0.5>10−1>10−1.50.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
Example 41
Alamar Blue Endothelial Cells Proliferation Assay
This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.
Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37 degrees C. overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of an albumin fusion protein of the invention or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C. incubator for three days. After three days 10 ml of stock alamar blue (Biosource Cat# DAL1100) is added to each well and the plate(s) is/are placed back in the 37° C. incubator for four hours. The plate(s) are then read at 530 nm excitation and 590 nm emission using the CytoFluor fluorescence reader. Direct output is recorded in relative fluorescence units.
Alamar blue is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form (i.e., stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity). The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.
Example 42
Detection of Inhibition of a Mixed Lymphocyte Reaction
This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by fusion proteins of the invention. Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by the albumin fusion proteins that inhibit MLR since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.
Albumin fusion proteins of the invention found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.
Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×106 cells/ml in RPMI-1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is adjusted to 2×105 cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of the fusion protein test material (50 μl) is added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO2, and 1 μC of [3H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.
Samples of the fusion protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.
Example 43
Assays for Protease Activity
The following assay may be used to assess protease activity of an albumin fusion protein of the invention.
Gelatin and casein zymography are performed essentially as described (Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are run on 10% polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in 2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3 at 37° C. 5 to 16 hours. After staining in amido black areas of proteolysis apear as clear areas agains the blue-black background. Trypsin (Sigma T8642) is used as a positive control.
Protease activity is also determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B4500. Reactions are set up in (25 mMNaPO4, 1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples are added and the change in adsorbance at 260 nm is monitored on the Beckman DU6 spectrophotometer in the time-drive mode. Trypsin is used as a positive control.
Additional assays based upon the release of acid-soluble peptides from casein or hemoglobin measured as adsorbance at 280 nm or calorimetrically using the Folin method are performed as described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assays involve the solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).
Example 44
Identifying Serine Protease Substrate Specificity
Methods known in the art or described herein may be used to determine the substrate specificity of the albumin fusion proteins of the present invention having serine protease activity. A preferred method of determining substrate specificity is by the use of positional scanning synthetic combinatorial libraries as described in GB 2 324 529 (incorporated herein in its entirety).
Example 45
Ligand Binding Assays
The following assay may be used to assess ligand binding activity of an albumin fusion protein of the invention.
Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for an albumin fusion protein of the invention is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards the fusion protein. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
Example 46
Functional Assay in Xenopus Oocytes 
Capped RNA transcripts from linearized plasmid templates encoding an albumin fusion protein of the invention is synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocytc) are injected in a 50 nl bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response fusion protein and polypeptide agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
Example 47
Microphysiometric Assays
Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of detecting the ability of an albumin fusion protein of the invention to activate secondary messengers that are coupled to an energy utilizing intracellular signaling pathway.
Example 48
Extract/Cell Supernatant Screening
A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligands banks as identified to date. Accordingly, the albumin fusion proteins of the invention can also be functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands for the Therapeutic protein portion and/or albumin protein portion of an albumin fusion protein of the invention. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
Example 49
ATP-binding Assay
The following assay may be used to assess ATP-binding activity of fusion proteins of the invention.
ATP-binding activity of an albumin fusion protein of the invention may be detected using the ATP-binding assay described in U.S. Pat. No. 5,858,719, which is herein incorprated by reference in its entirety. Briefly, ATP-binding to an albumin fusion protein of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay. Reaction mixtures containing 1 mg/ml of ABC transport protein are incubated with varying concentrations of ATP, or the non-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at 4° C. A mixture of 8-azido-ATP (Sigma Chem. Corp., St Louis, Mo.) plus 8-azido-ATP (32P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to a final concentration of 100 μM and 0.5 ml aliquots are placed in the wells of a porcelain spot plate on ice. The plate is irradiated using a short wave 254 nm UV lamp at a distance of 2.5 cm from the plate for two one-minute intervals with a one-minute cooling interval in between. The reaction is stopped by addition of dithiothreitol to a final concentration of 2 mM. The incubations are subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. Protein bands corresponding to the albumin fusion proteins of the invention are excised, and the radioactivity quantified. A decrease in radioactivity with increasing ATP or adenly-5′-imidodiphosphate provides a measure of ATP affinity to the fusion protein.
Example 50
Phosphorylation Assay
In order to assay for phosphorylation activity of an albumin fusion protein of the invention, a phosphorylation assay as described in U.S. Pat. No. 5,958,405 (which is herein incorporated by reference) is utilized. Briefly, phosphorylation activity may be measured by phosphorylation of a protein substrate using gamma-labeled 32P-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. The fusion portein of the invention is incubated with the protein substrate, 32P-ATP, and a kinase buffer. The 32P incorporated into the substrate is then separated from free 32P-ATP by electrophoresis, and the incorporated 32P is counted and compared to a negative control. Radioactivity counts above the negative control are indicative of phosphorylation activity of the fusion protein.
Example 51
Detection of Phosphorylation Activity (Activation) of an Albumin Fusion Protein of the Invention in the Presence of Polypeptide Ligands
Methods known in the art or described herein may be used to determine the phosphorylation activity of an albumin fusion protein of the invention. A preferred method of determining phosphorylation activity is by the use of the tyrosine phosphorylation assay as described in U.S. Pat. No. 5,817,471 (incorporated herein by reference).
Example 52
Identification of Signal Transduction Proteins that Interact with an Albumin Fusion Protein of the Present Invention
Albumin fusion proteins of the invention may serve as research tools for the identification, characterization and purification of signal transduction pathway proteins or receptor proteins. Briefly, a labeled fusion protein of the invention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, an albumin fusion protein of the invention is covalenty coupled to a chromatography column. Cell-free extract derived from putative target cells, such as carcinoma tissues, is passed over the column, and molecules with appropriate affinity bind to the albumin fusion protein. The protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 53
IL-6 Bioassay
A variety of assays are known in the art for testing the proliferative effects of an albumin fusion protein of the invention. For example, one such assay is the IL6 Bioassay as described by Marz et al. (Proc. Natl. Acad. Sci., U.S.A., 95:3251-56 (1998), which is herein incorporated by reference). After 68 hrs. at 37° C., the number of viable cells is measured by adding the tetrazolium salt thiazolyl blue (MTT) and incubating for a further 4 hrs. at 37° C. B9 cells are lysed by SDS and optical density is measured at 570 nm. Controls containing IL6 (positive) and no cytokine (negative) are Briefly, IL-6 dependent B9 murine cells are washed three times in IL-6 free medium and plated at a concentration of 5,000 cells per well in 50 μl, and 50 μl of fusion protein of the invention is added, utilized. Enhanced proliferation in the test sample(s) (containing an albumin fusion protein of the invention) relative to the negative control is indicative of proliferative effects mediated by the fusion protein.
Example 54
Support of Chicken Embryo Neuron Survival
To test whether sympathetic neuronal cell viability is supported by an albumin fusion protein of the invention, the chicken embryo neuronal survival assay of Senaldi et al may be utilized (Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998), which is herein incorporated by reference). Briefly, motor and sympathetic neurons are isolated from chicken embryos, resuspended in L15 medium (with 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; Life Technologies, Rockville, Md.) and Dulbecco's modified Eagles medium [with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2); Life Technologies, Rockville, Md.], respectively, and incubated at 37° C. in 5% CO2 in the presence of different concentrations of the purified fusion protein of the invention, as well as a negative control lacking any cytokine. After 3 days, neuron survival is determined by evaluation of cellular morphology, and through the use of the colorinmetric assay of Mosmann (Mosmann; T., J. Inmnunol. Methods, 65:55-63 (1983)). Enhanced neuronal cell viability as compared to the controls lacking cytokine is indicative of the ability of the albumin fusion protein to enhance the survival of neuronal cells.
Example 55
Assay for Phosphatase Activity
The following assay may be used to assess serine/threonine phosphatase (PTPase) activity of an albumin fusion protein of the invention.
In order to assay for serine/threonine phosphatase (PTPase) activity, assays can be utilized which are widely known to those skilled in the art. For example, the serine/threonine phosphatase (PSPase) activity of an albumin fusion protein of the invention may be measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine residues with cAMP-dependent Protein Kinase in the presence of [32P]ATP. Protein serine/threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.
Example 56
Interaction of Serine/Threonine Phosphatases with Other Proteins
Fusion protein of the invention having serine/threonine phosphatase activity (e.g., as determined in Example 55) are useful, for example, as research tools for the identification, characterization and purification of additional interacting proteins or receptor proteins, or other signal transduction pathway proteins. Briefly, a labeled fusion protein of the invention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, an albumin fusion protein of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as neural or liver cells, is passed over the column, and molecules with appropriate affinity bind to the fusion protein. The fusion protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 57
Assaying for Heparanase Activity
There a numerous assays known in the art that may be employed to assay for heparanase activity of an albumin fusion protein of the invention. In one example, heparanase activity of an albumin fusion protein of the invention, is assayed as described by Vlodavsky et al., (Vlodavsky et al., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditioned media, intact cells (1×106 cells per 35-mm dish), cell culture supernatant, or purified fusion protein are incubated for 18 hrs at 37° C., pH 6.2-6.6, with 35S-labeled ECM or soluble ECM derived peak 1 proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a Sepharose CL-6B column (0.9×30 cm). Fractions are eluted with PBS and their radioactivity is measured. Degradation fragments of heparan sulfate side chains are eluted from Sepharose 6B at 0.5<Kav<0.8 (peak II). Each experiment is done at least three times. Degradation fragments corresponding to “peak II,” as described by Vlodavsky et al., is indicative of the activity of an albumin fusion protein of the invention in cleaving heparan sulfate.
Example 58
Immobilization of Biomolecules
This example provides a method for the stabilization of an albumin fusion protein of the invention in non-host cell lipid bilayer constucts (see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999), hereby incorporated by reference in its entirety herein) which can be adapted for the study of fusion proteins of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine biotin tag to an albumin fusion protein of the invention, thus allowing uniform orientation upon immobilization. A 50 uM solution of an albumin fusion protein of the invention in washed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150 ul). Then the sample is dialyzed (Pierce Slidealizer Cassett; 10 kDa cutoff; Pierce Chemical Co., Roclkford Ill.) at 4C first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 59
Assays for Metalloproteinase Activity
Metalloproteinases are peptide hydrolases which use metal ions, such as Zn2+, as the catalytic mechanism. Metalloproteinase activity of an albumin fusion protein of the present invention can be assayed according to methods known in the art. The following exemplary methods are provided:
Proteolysis of Alpha-2-macroglobulin
To confirm protease activity, a purified fusion protein of the invention is mixed with the substrate alpha-2-macroglobulin (0.2 unit/ml; Boehringer Mannheim, Germany) in 1×assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl2, 25 μM ZnCl2 and 0.05% Brij-35) and incubated at 37° C. for 1-5 days. Trypsin is used as positive control. Negative controls contain only alpha-2-macroglobulin in assay buffer. The samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresis the proteins are visualized by silver staining. Proteolysis is evident by the appearance of lower molecular weight bands as compared to the negative control.
Inhibition of Alpha-2-macroglobulin Proteolysis by Inhibitors of Metalloproteinases
Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, AND HgCl2), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2), and commercial small molecule MMP inhibitors) may also be used to characterize the proteolytic activity of an albumin fusion protein of the invention. Three synthetic MMP inhibitors that may be used are: MMP inhibitor I, [IC50=1.0 μM against MMP-1 and MMP-8; IC50=30 μM against MMP-9; IC50=150 μM against MMP-3]; MMP-3 (stromelysin-1) inhibitor I [IC50=5 μM against MMP-3], and MMP-3 inhibitor II (Ki=130 nM against MMP-3]; inhibitors available through Calbiochem, catalog # 444250, 444218, and 444225, respectively). Briefly, different concentrations of the small molecule MMP inhibitors are mixed with a purified fusion protein of the invention (50 μg/ml) in 22.9 μl of 1×HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl2, 25 μM ZnCl2 and 0.05%Brij-35) and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl of substrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at 37° C. for 20-hr. The reactions are stopped by adding 4× sample buffer and boiled immediately for 5 minutes. After SDS-PAGE, the protein bands are visualized by silver stain.
Synthetic Fluorogenic Peptide Substrates Cleavage Assay
The substrate specificity for fusion proteins of the invention with demonstrated metalloproteinase activity may be determined using techniques knonw in the art, such as using synthetic fluorogenic peptide substrates (purchased from BACHEM Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four are MMP substrates and the last one is a substrate of tumor necrosis factor-α (TNF-α) converting enzyme (TACE). These substrastes are preferably prepared in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are 50-500 μM. Fluorescent assays are performed by using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nm. Briefly, the assay is carried out by incubating 176 μl 1×HEPES buffer (0.2 M NaCl, 10 MM CaCl2, 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, and then adding 20 μl of a purified fusion protein of the invention into the assay cuvett. The final concentration of substrate is 1 μM. Initial hydrolysis rates are monitored for 30-min.
Example 60
Identification and Cloning of VH and VL Domains
One method to identfy and clone VH and VL domains from cell lines expressing a particular antibody is to perform PCR with VH and VL specific primers on cDNA made from the antibody expressing cell lines. Briefly, RNA is isolated from the cell lines and used as a template for RT-PCR designed to amplify the VH and VL domains of the antibodies expressed by the EBV cell lines. Cells may be lysed in the TRIzol® reagent (Life Technologies, Rockville. Md.) and extracted with one fifth volume of chloroform. After addition of chloroform, the solution is allowed to incubate at room temperature for 10 minutes, and the centrifuged at 14,000 rpm for 15 minutes at 4° C. in a tabletop centrifuge. The supernatant is collected and RNA is precipitated using an equal volume of isopropanol. Precipitated RNA is pelleted by centrifuging at 14,000 rpm for 15 minutes at 4° C. in a tabletop centrifuge. Following centrifugation, the supernatant is discarded and washed with 75% ethanol. Follwing washing, the RNA is centrifuged again at 800 rpm for 5 minutes at 4° C. The supernatant is discarded and the pellet allowed to air dry. RNA is the dissolved in DEPC water and heated to 60° C. for 10 minutes. Quantities of RNA can determined using optical density measurements.
cDNA may be synthesized, according to methods well-known in the art, from 1.5-2.5 micrograms of RNA using reverse transciptase and random hexamer primers. cDNA is then used as a template for PCR amplification of VH and VL domains. Primers used to amplify VH and VL genes are shown in Table 3. Typically a PCR reaction makes use of a single 5′ primer and a single 3′ primer. Sometimes, when the amount of available RNA template is limiting, or for greater efficiency, groups of 5′ and/or 3′ primers may be used. For example, sometimes all five VH-5′ primers and all JH3′ primers are used in a single PCR reaction. The PCR reaction is carried out in a 50 microliter volume containing 1×PCR buffer, 2 mM of each dNTP, 0.7 units of High Fidelity Taq polymerse, 5′ primer mix, 3′ primer mix and 7.5 microliters of cDNA. The 5′ and 3′ primer mix of both VH and VL can be made by pooling together 22 pmole and 28 pmole, respectively, of each of the individual primers. PCR conditions are: 96° C. for 5 minutes; followed by 25 cycles of 94° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 1 minute; followed by anextension cycle of 72° C. for 10 minutes. After the reaction is completed, sample tubes are stored 4° C.







TABLE 3







Primer Sequences Used to Amplify VH and VL domains.












SEQ
Primer



Primer
ID
Sequence



name
NO
(5′-3′)







VH Primers





Hu VH1-5′
36
CAGGTGCAGCTGGTGCAGTCTGG



Hu VH2-5′
37
CAGGTCAACTTAAGGGAGTCTGG



Hu VH3-5′
38
GAGGTGCAGCTGGTGGAGTCTGG



Hu VH4-5′
39
CAGGTGCAGCTGCAGGAGTCGGG



Hu VH5-5′
40
GAGGTGCAGCTGTTGCAGTCTGC



Hu VH6-5′
41
CAGGTACAGCTGCAGCAGTCAGG



Hu JH1,2-5′
42
TGAGGAGACGGTGACCAGGGTGCC



Hu JH3-5′
43
TGAAGAGACGGTGACCAftGTCCC



Hu JH4,5-5′
44
TGAGGAGACGGTGACCAGGGTFCC



Hu JH6-5′
45
TGAGGAGACGGTGACCGTGGTCCC



VL Primers



Hu Vkappa1-5′
46
GACATCCAGATGACCCAGTCTCC



Hu Vkappa2a-5′
47
GATGTGTGATGACTCAGTCTCC



Hu Vkappa2b-5′
48
GATATTGTGATGACTCAGTCTCC



Hu Vkappa3-5′
49
GAAATTGT6TTGACGCAGTCTCC



Hu Vkappa4-5′
50
GACATCGTGATGACCCAGTCTCC



Hu Vkappa5-5′
51
GAAACGACACTCACGCAGTCTCC



Hu Vkappa6-5′
52
GAAATTGTGCTGACTCAGTCTCC



Hu Vlambda1-5′
53
CAGTCTGTGTTGACGCAGCCGCC



Hu Vlambda2-5′
54
CAGTCTGCCCTGACTCAGCCTGC



Hu Vlambda3-5′
55
TCCTATGTGCTGACTCAGCCACC



Hu Vlambda3b-5′
56
TCTTCTGAGCTGACTCAGGACCC



Hu Vlambda4-5′
57
CACGTTATACTGACTCAACCGCC



Hu Vlambda5-5′
58
CAGGCTGTGCTCACTCAGCCGTC



Hu Vlambda6-5′
59
AATTTTATGCTGACTCAGCCCCA



Hu Jkappa1-3′
60
ACGTTTGATTCCACCTTGGTCCC



Hu Jkappa2-3′
61
ACGTTTGATCTCCAGCTTGGTCCC



Hu Jkappa3-3′
62
ACGTTTGATATCCACTTTGGTCCC



Hu Jkappa4-3′
63
ACGTTTGATCTCCACCTTGGTCCC



Hu Jkappas5-3′
64
ACGTTTAATCTCCAGTCGTGTCCC



Hu Jlambda1-3′
65
CAGTCTGTGTTGACGCAGCCGCC



Hu Jlambda2-3′
66
CAGTCTGCCCTGACTCAGCCTGC



Hu Jlambda3--3′
67
TCCTATGTGCTGACTCAGCCACC



Hu Jlambda3b-3′
68
TCTTCTGAGCTGACTCAGGACCC



Hu Jlambda4-3′
69
CACGTTATACTGACTCAACCGCC



Hu Jlambda5-3′
70
CAGGCTGTGCTCACTCAGCCGTC



Hu Jlambda6-3′
71
AATFTTATGCTGACTCAGCCCCA










PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands of the expected sizes ( ̃506 base pairs for VH domains, and 344 base pairs for VL domains) can be cut out of the gel and purified using methods well known in the art. Purified PCR products can be ligated into a PCR cloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.). Individual cloned PCR products can be isolated after transfection of E. coli and blue/white color selection. Cloned PCR products may then be sequenced using methods commonly known in the art.
The PCR bands containing the VH domain and the VL domains can also be used to create full-length Ig expression vectors. VH and VL domains can be cloned into vectors containing the nucleotide sequences of a heavy (e.g., human IgG1 or human IgG4) or light chain (human kappa or human lambda) constant regions such that a complete heavy or light chain molecule could be expressed from these vectors when transfected into an appropriate host cell. Further, when cloned heavy and light chains are both expressed in one cell line (from either one or two vectors), they can assemble into a complete functional antibody molecule that is secreted into the cell culture medium. Methods using polynucleotides encoding VH and VL antibody domain to generate expression vectors that encode complete antibody molecules are well known within the art.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent applications, patent publications, journal articles, abstracts, laboratory manuals, books, or other disclosures) as well as information available through Identifiers specific to databases such as GenBank, GeneSeq, or the CAS Registry, referred to in this application are herein incorporated by reference in their entirety. The specification and sequence listing of each of the following U.S. applications are herein incorporated by reference in their entirety: application Ser. Nos. 09/091,873 filed Jun. 25, 1998; 60/229,358 filed on Apr. 12, 2000; 60/199,384 filed on Apr. 25, 2000 and 60/256,931 filed on Dec. 21, 2000.

## Claims

1. An albumin fusion protein comprising a member selected from the group consisting of:
(a) a Therapeutic protein X and albumin comprising the amino acid sequence of SEQ ID NO:18; 
(b) a Therapeutic protein X and a fragment or a variant of the amino acid sequence of SEQ ID NO:18, wherein said fragment or variant has albumin activity; 
(c) a Therapeutic protein X and a fragment or a variant of the amino acid sequence of SEQ ID NO:18, wherein said fragment or variant has albumin activity, and further wherein said albumin activity is the ability to prolong the shelf life of the Therapeutic protein X compared to the shelf-life of the Therapeutic protein X in an unfused state; 
(d) a Therapeutic protein X and a fragment or a variant of the amino acid sequence of SEQ ID NO:18, wherein said fragment or variant has albumin activity, and further wherein the fragment or variant comprises the amino acid sequence of amino acids 1-387 of SEQ ID NO:18; 
(e) a fragment or variant of a Therapeutic protein X and albumin comprising the amino acid sequence of SEQ ID NO:18, wherein said fragment or variant has a biological activity of the Therapeutic protein X; 
(f) a Therapeutic protein X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (e), wherein the Therapeutic protein X, or fragment or variant thereof, is fused to the N-terminus of albumin, or the N-terminus of the fragment or variant of albumin; 
(g) a Therapeutic protein X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (e), wherein the Therapeutic protein X, or fragment or variant thereof, is fused to the C-terminus of albumin, or the C-terminus of the fragment or variant of albumin; 
(h) a Therapeutic protein X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (e), wherein the Therapeutic protein X, or fragment or variant thereof, is fused to the N-terminus and C-terminus of albumin, or the N-terminus and the C-terminus of the fragment or variant of albumin; 
(i) a Therapeutic protein X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (e), which comprises a first Therapeutic protein X, or fragment or variant thereof, and a second Therapeutic protein X, or fragment or variant thereof, wherein said first Therapeutic protein X, or fragment or variant thereof, is different from said second Therapeutic protein X, or fragment or variant thereof; 
(j) a Therapeutic protein X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (i), wherein the Therapeutic protein X, or fragment or variant thereof, is separated from the albumin or the fragment or variant of albumin by a linker; and 
(k) a Therapeutic protein X, or fragment or variant thereof, and albumin, or fragment or variant thereof, of (a) to (j), wherein the albumin fusion protein has the following formula: 


R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, 

and further wherein R1 is Therapeutic protein X, or fragment or variant thereof, L is a peptide linker, and R2 is albumin comprising the amino acid sequence of SEQ ID NO:18 or a fragment or variant of albumin, 
wherein the Therapeutic protein X is selected from at least one of the proteins set forth in Table 1.

2. The albumin fusion protein of claim 1, wherein the shelf-life of the albumin fusion protein is greater than the shelf-life of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

3. The albumin fusion protein of claim 1, wherein the in vitro biological activity of the Therapeutic protein X, or fragment or variant thereof, fused to albumin, or fragment or variant thereof, is greater than the in vitro biological activity of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

4. The albumin fusion protein of claim 1, wherein the in vivo biological activity of the Therapeutic protein X, or fragment or variant thereof, fused to albumin, or fragment or variant thereof, is greater than the in vivo biological activity of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

5. An albumin fusion protein comprising a Therapeutic protein X, or fragment or variant thereof, inserted into an albumin, or fragment or variant thereof, comprising the amino acid sequence of SEQ ID NO:18 or fragment or variant thereof, wherein the Therapeutic Protein X is selected from at least one of the proteins set forth in Table 1.

6. An albumin fusion protein comprising a Therapeutic protein X, or fragment or variant thereof, inserted into an albumin, or fragment or variant thereof, comprising an amino acid sequence selected from the group consisting of:
(a) amino acids 54 to 61 of SEQ ID NO:18; 
(b) amino acids 76 to 89 of SEQ ID NO:18; 
(c) amino acids 92 to 100 of SEQ ID NO:18; 
(d) amino acids 170 to 176 of SEQ ID NO:18; 
(e) amino acids 247 to 252 of SEQ ID NO:18; 
(f) amino acids 266 to 277 of SEQ ID NO:18; 
(g) amino acids 280 to 288 of SEQ ID NO:18; 
(h) amino acids 362 to 368 of SEQ ID NO:18; 
(i) amino acids 439 to 447 of SEQ ID NO:18; 
(j) amino acids 462 to 475 of SEQ ID NO:18; 
(k) amino acids 478 to 486 of SEQ ID NO:18; and 
(l) amino acids 560 to 566 of SEQ ID NO:18; 

wherein the Therapeutic protein X is selected from at least one of the proteins set forth in Table 1.

7. The albumin fusion protein of claim 5, wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the shelf-life of the Therapeutic protein X, or fragment or variant thereof, as compared to the shelf-life of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

8. The albumin fusion protein of claim 6, wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the shelf-life of the Therapeutic protein X, or fragment or variant thereof, as compared to the shelf-life of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

9. The albumin fusion protein of claim 5, wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the in vitro biological activity of the Therapeutic protein X, or fragment or variant thereof, fused to albumin as compared to the in vitro biological activity of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

10. The albumin fusion protein of claim 6, wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the in vitro biological activity of the Therapeutic protein X, or fragment or variant thereof, fused to albumin as compared to the in vitro biological activity of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

11. The albumin fusion protein of claim 5 wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the in vivo biological activity of the Therapeutic protein X, or fragment or variant thereof, fused to albumin compared to the in vivo biological activity of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

12. The albumin fusion protein of claim 6 wherein said albumin fusion protein comprises a portion of albumin sufficient to prolong the in vivo biological activity of the Therapeutic protein X, or fragment or variant thereof, fused to albumin compared to the in vivo biological activity of the Therapeutic protein X, or fragment or variant thereof, in an unfused state.

13. The albumin fusion protein of any one of claims 1-12, which is non-glycosylated.

14. The albumin fusion protein of any one of claims 1-12, which is expressed in yeast.

15. The albumin fusion protein of claim 14, wherein the yeast is glycosylation deficient.

16. The albumin fusion protein of claim 14 wherein the yeast is glycosylation and protease deficient.

17. The albumin fusion protein of any one of claims 1-12, which is expressed by a mammalian cell.

18. The albumin fusion protein of any one of claims 1-12, wherein the albumin fusion protein is expressed by a mammalian cell in culture.

19. The albumin fusion protein of any one of claims 1-12, wherein the albumin fusion protein further comprises a secretion leader sequence.

20. A composition comprising the albumin fusion protein of any one of claims 1-12 and a pharmaceutically acceptable carrier.

21. A kit comprising the composition of claim 20.

22. A method of extending the shelf life of Therapeutic protein:X, or fragment or variant thereof, comprising the step of fusing the Therapeutic protein:X, or fragment or variant thereof, to albumin, or fragment or variant thereof, sufficient to extend the shelf-life of the Therapeutic protein:X, or fragment or variant thereof, compared to the shelf-life of the Therapeutic protein:X, or fragment or variant thereof, in an unfused state.

23. A nucleic acid molecule comprising a polynucleotide sequence encoding the albumin fusion protein of any one of claims 1-12.

24. A vector comprising the nucleic acid molecule of claim 23.

25. A host cell comprising the nucleic acid molecule of claim 24.