# Patent

## Title
Antagonist anti-Notch3 antibodies and their use in the prevention and treatment of Notch3-related diseases

## Abstract

The present invention relates to antagonist antibodies that specifically bind to Notch 3 and inhibit its activation. The present invention includes antibodies binding to a conformational epitope comprising the first Lin12 domain and the second dimerization domain. The present invention also includes uses of these antibodies to treat or prevent Notch 3 related diseases or disorders.

## Background
RELATED APPLICATIONS
This application is a divisional application of U.S. patent application Ser. No. 11/958,099, filed Dec. 17, 2007, now U.S. Pat. No. 7,935,791, which claims the benefit of U.S. Provisional Patent Application No. 60/875,597, filed Dec. 18, 2006, and U.S. Provisional Patent Application No. 60/879,218, filed Jan. 6, 2007. The disclosures of the foregoing applications are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to antagonist anti-Notch3 antibodies and their use in the amelioration, treatment, or prevention of a Notch3-related disease or disorder.
BACKGROUND OF THE INVENTION
The Notch gene was first described in 1917 when a strain of the fruit fly Drosophila melanogaster was found to have notched wing blades (Morgan, Am Nat 51:513 (1917)). The gene was cloned almost seventy years later and was determined to be a cell surface receptor playing a key role in the development of many different cell types and tissues in Drosophila (Wharton et al., Cell 43:567 (1985)). The Notch signaling pathway was soon found to be a signaling mechanism mediated by cell-cell contact and has been evolutionarily conserved from Drosophila to human. Notch receptors have been found to be involved in many cellular processes, such as differentiation, cell fate decisions, maintenance of stem cells, cell motility, proliferation, and apoptosis in various cell types during development and tissue homeostasis (For review, see Artavanis-Tsakonas, et al., Science 268:225 (1995)).
Mammals possess four Notch receptor proteins (designated Notch1 to Notch4) and five corresponding ligands (designated Delta-1 (DLL-1), Delta-3 (DLL-3), Delta-4 (DLL-4), Jagged-1 and Jagged-2). The mammalian Notch receptor genes encode  ̃300 kD proteins that are cleaved during their transport to the cell surface and exist as heterodimers. The extracellular portion of the Notch receptor has thirty-four epidermal growth factor (EGF)-like repeats and three cysteine-rich Notch/LIN12 repeats. The association of two cleaved subunits is mediated by sequences lying immediately N-terminal and C-terminal of the cleavage site, and these two subunits constitute the Notch heterodimerization (HD) domains (Wharton, et al., Cell 43:567 (1985); Kidd, et al., Mol Cell Biol 6:3431 (1986); Kopczynski, et al., Genes Dev 2:1723 (1988); Yochem, et al., Nature 335:547 (1988)).
At present, it is still not clear how Notch signaling is regulated by different receptors or how the five ligands differ in their signaling or regulation. The differences in signaling and/or regulation may be controlled by their expression patterns in different tissues or by different environmental cues. It has been documented that Notch ligand proteins, including Jagged/Serrate and Delta/Delta-like, specifically bind to the EGF repeat region and induce receptor-mediated Notch signaling (reviewed by Bray, Nature Rev Mol Cell Biol. 7:678 (2006), and by Kadesch, Exp Cell Res. 260:1 (2000)). Among the EGF repeats, the 10th to 12th repeats are required for ligand binding to the Notch receptor, and the other EGF repeats may enhance receptor-ligand interaction (Xu, et al., J Biol Chem. 280:30158 (2005); Shimizu, et al., Biochem Biophys Res Comm. 276:385 (2000)). Although the LIN12 repeats and the dimerization domain are not directly involved in ligand binding, they play important roles in maintaining the heterodimeric protein complex, preventing ligand-independent protease cleavage and receptor activation (Sanche-Irizarry, et al., Mol Cell Biol. 24:9265 (2004); Vardar et al., Biochem. 42:7061 (2003)).
The expression of mutant forms of Notch receptors in developing Xenopus embryos interferes profoundly with normal development (Coffman, et al., Cell 73: 659 (1993)). A Notch1 knockout was found to be embryonic lethal in mice (Swiatek, et al., Genes & Dev 8:707 (1994)). In humans, there have been several genetic diseases, including cancer, linked to different Notch receptor mutations (Artavanis-Tsakonas, et al., Science 284:770 (1999)). For instance, aberrant activation of Notch1 receptor caused by translocation can lead to T cell lymphoblastic leukemia (Ellisen, et al., Cell 66:649 (1991)). Certain mutations in the HD domains of Notch1 receptor enhance signaling without ligand binding (Malecki, et al., Mol Cell Biol 26:4642 (2006)), further implicating their roles in Notch receptor activation. The signal induced by ligand binding is transmitted to the nucleus by a process involving two proteolytic cleavages of the receptor followed by nuclear translocation of the intracellular domain (Notch-IC). Although LIN12 repeats and HD domains were thought to prevent signaling in the absence of ligands, it is still unclear how ligand binding facilitates proteolytic cleavage events.
Notch receptors have been linked to a wide range of diseases including cancer, neurological disorders, and immune diseases, as evidenced by reports of the over-expression of Notch receptors in various human disease tissues and cell lines as compared to normal or nonmalignant cells (Joutel, et al. Cell & Dev Biol 9:619 (1998); Nam, et al., Curr Opin Chem Biol 6:501 (2002)). The Notch3 receptor is over-expressed in various solid tumors, including non-small cell lung cancer (NSCLC) and ovarian cancer (Haruki, et al., Cancer Res 65:3555 (2005); Park, et al., Cancer Res 66:6312 (2006); Lu, et al., Clin Cancer Res 10:3291 (2004)), suggesting the significance of Notch3 receptor expression in solid tumors. Furthermore, Notch3 receptor expression is upregulated in plasma cell neoplasms, including multiple myeloma, plasma cell leukemia, and extramedullary plasmacytoma (Hedvat, et al., Br J Haematol 122:728 (2003); pancreatic cancer (Buchler, et al., Ann Surg 242:791 (2005)); and T cell acute lymphoblastic leukemias (T-ALL) (Bellavia, et al., Proc Natl Acad Sci USA 99:3788 (2002); Screpanti, et al., Trends Mol Med 9:30 (2003)). Notch3 receptor is also expressed in a subset of neuroblastoma cell lines and serves as a marker for this type of tumor that has constitutional or tumor-specific mutations in the homeobox gene Phox2B (van Limpt, et al., Cancer Lett 228:59 (2005)). Other indications and diseases that have been linked to Notch3 receptor expression include neurological disorders (Joutel, et al., Nature 383:707 (1996)), diabetes (Anastasi, et al., J Immunol 171:4504 (2003), rheumatoid arthritis (Yabe, et al., J Orthop Sci 10:589 (2005)), vascular related diseases (Sweeney, et al., FASEB J 18:1421 (2004)), and Alagille syndrome (Flynn, et al., J Pathol 204:55 (2004)).
Although Notch3 receptor over-expression (including gene amplification) has been observed in various cancers, no activating mutations have yet been reported. It is plausible that an increased level of Notch3 receptors in tumors can be activated by different ligands in stromal cells or tumor cells and lead to enhanced Notch3 signaling. Particularly, Notch ligands have been localized to the vascular endothelium during both development and tumorigenesis (Mailhos, et al., Differentiation 69:135 (2001); Taichman, et al., Dev Dyn 225: 166 (2002)), suggesting endothelial cells could provide the ligands for Notch3 receptor activation in tumors. Similar tumor-stroma cross-talk mediated by Notch ligand and receptor have been demonstrated in different type of cancers (Houde, et al., Blood 104: 3697 (2004); Jundt, et al., Blood 103: 3511 (2004); Zeng, et al., Cancer Cell 8: 13 (2005)). Increased Notch3 signaling caused by over-expression of intracellular Notch3 (Notch3-IC) can lead to tumorigenesis in T-ALL and breast cancer animal models (Vacca, et al., The EMBO J 25: 1000 (2006); Hu, et al., Am J Pathol 168: 973 (2006)).
Notch signaling and its role in cell self-renewal have been implicated in cancer stem cells, which are a minority population in tumors and can initiate tumor formation (Reya, et al., Nature 414:105 (2001)). Normal stem cells from many tissues, including intestinal and neuronal stem cells, depend on Notch signaling for self-renewal and fate determination (Fre, et al., Nature, 435: 964 (2005); van Es, et al., Nature, 435:959 (2005); Androutsellis-Theotokis, et al., Nature, 442: 823 (2006)). Similar mechanisms could exist in cancer stem cells, and inhibition of Notch signaling by γ-secretase inhibitors was shown to deplete cancer stem cells and block engraftment in embryonal brain tumors (Fan, et al., Cancer Res 66:7445 (2006)).
Inhibition of Notch signaling by γ-secretase inhibitor has striking antineoplastic effects in Notch-expressing transformed cells in vitro and in xenograft models (Weijzen, et al., Nat Medicine 8: 879 (2002); Bocchetta, et al., Oncogene 22:81 (2003); Weng, et al., Science, 306:269 (2004)). More recently, a γ-secretase inhibitor has been shown to efficaciously kill colon adenomas in Apc (min+) mice (van Es, et al., Nature, 435: 959 (2005)), although the therapeutic window, due to its effect on normal stem cells and the inhibition of multiple Notch pathways, is very narrow. Different from Notch1, a Notch3 gene knockout in mice was not embryonically lethal and had few defects (Domenga, et al., Genes & Dev 18: 2730 (2004)), suggesting that Notch 3 provides a potentially better therapeutic target than Notch 1.
Tournier-Lasserve et al. (U.S. Application 2003/0186290) teach the association of Notch3 receptor and CADASIL. The application discloses various mutations in the Notch3 gene and their possible association with the disease CADASIL. The application suggests the use of diagnostic antibodies to detect such mutations. The application also suggests therapeutic antibodies to treat CADASIL, i.e. agonistic antibodies, but no specific antibodies are disclosed nor how to make such antibodies.
In view of the large number of human diseases associated with the Notch3 signaling pathway, it is important that new ways of preventing and treating these diseases be identified. The current invention provides novel anti-Notch3 antibodies useful for this unmet medical need.
SUMMARY OF THE INVENTION
The present invention provides novel antibodies and fragments thereof that specifically bind to a conformational epitope of the human Notch3 receptor, the epitope comprising the LIN12 domain and the heterodimerization domain. Another aspect of the invention includes the epitope binding site and antibodies that bind this same epitope as the antibodies of the present invention. The antibodies of the present invention inhibit ligand-induced signaling through the Notch3 receptor.
The invention includes the amino acid sequences of the variable heavy and light chain of the antibodies and their corresponding nucleic acid sequences. Another embodiment of the invention includes the CDR sequences of these antibodies. Another embodiment includes humanized forms of these antibodies.
Another embodiment of the present invention includes the cell lines and vectors harboring the antibody sequences of the present invention.
The present invention also includes the conformational epitope recognized by the antagonist antibodies of the invention. The present invention also includes antibodies that bind this conformational epitope. The embodiments include a Notch 3 conformational epitope comprising the LIN12 domain having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 9 and the dimerization domain 2 having at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 18. More particularly, the Notch 3 conformational epitope comprising amino acid residues 1395-1396, 1402-1404 and 1420-1422 of the L1 LIN12 domain and amino acid residues 1576-1578 and 1626-1628 of the D2 dimerization domain. The present invention includes antibodies that bind this conformational epitope.
Another embodiment of the preset invention is the use of any of these antibodies for the preparation of a medicament or composition for the treatment of diseases and disorders associated with Notch 3 receptor activation.
Another embodiment of the preset invention is the use of any of these antibodies in the treatment of disorders associated with Notch 3 activation comprising the inhibition of said activation by, e.g., inhibiting Notch 3 signaling, or neutralization of the receptor by blocking ligand binding. Notch 3 related disorders may include, but are not limited to, T-cell acute lymphoblastic leukemia, lymphoma, liver disease involving aberrant vascularization, diabetes, ovarian cancer, diseases involving vascular cell fate, rheumatoid arthritis, pancreatic cancer, non-small cell lung cancer, plasma cell neoplasms (such as multiple myeloma, plasma cell leukemia, and extramedullary plasmacytoma), and neuroblastoma.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts the amino acid sequence of Notch3. The EGF repeat region extends from amino acid residue 43 to 1383; the LIN12 domain extends from amino acid residue 1384 to 1503; and the dimerization domain extends from amino acid residue 1504 to 1640.
FIG. 2 (A-H) depicts the amino acid sequence comparison between human Notch 1 (SEQ ID NO:44), Notch 2 (SEQ ID NO:45), Notch 3 (SEQ ID NO:1), and Notch 4 (SEQ ID NO:46).
FIG. 3 depicts the percent identity of Notch 1, Notch 2, Notch 3, and Notch 4.
FIGS. 4A and 4B depict the heavy and light chain variable region sequences of anti-Notch3 monoclonal antibody MAb 256A-4 (SEQ ID NO:2 and SEQ ID NO:3, respectively), with CDR regions underlined.
FIGS. 5A and 5B depict the heavy and light chain variable region sequences of anti-Notch3 monoclonal antibody MAb 256A-8 (SEQ ID NO: 4 and SEQ ID NO:5, respectively), with CDR regions underlined.
FIG. 6 depicts a luciferase reporter assay of Example 5 showing inhibitory effects by anti-Notch3 MAbs on the Notch3 ligand Jagged 1.
FIG. 7 depicts the luciferase reporter assay showing inhibitory effects by anti-Notch3 MAbs on the Notch3 ligand Jagged 2.
FIG. 8 depicts the luciferase reporter assay showing inhibitory effects by anti-Notch3 MAbs on the Notch3 ligand DLL4.
FIG. 9 depicts the luciferase reporter assay showing inhibitory effects to native Notch3 in ovarian cancer cells by anti-Notch3 MAbs. (9A) Human ovarian cancer cell line, OV/CAR3 and (9B) Human ovarian cancer cell line, A2780.
FIG. 10 depicts the apoptosis assay of Example 6 showing that cell survival effect induced by Jagged1 was inhibited by anti-Notch3 MAbs.
FIG. 11 depicts the inhibitory effect of anti-Notch3 MAbs on cell migration (11A) and invasion (11B) of Example 7.
FIG. 12 depicts a schematic diagram of the Notch1-Notch3 domain-swap protein expressed as a fusion protein with human IgG/Fc linked to C-terminus.
FIG. 13A depicts an ELISA using anti-human Fc control antibody as the detection antibody showing that the proteins of FIG. 12 were expressed in conditioned medium. FIG. 13B depicts an ELISA using 256A-4 as the detection antibody. FIG. 13C depicts an ELISA using 256A-8 as the detection antibody. FIG. 13D depicts an ELISA using a positive control antibody 256A-13 as the detection antibody.
FIG. 14 depicts the comparison of the engineered Notch3 leader peptide coding sequence (SEQ ID NO:47) to the native Notch3 leader peptide coding sequence (SEQ ID NO:48) (NCBI GENBANK® Accession No. NM—000435) showing the changes of nucleotides (14A) and the translated amino acid sequence of the engineered Notch leader peptide sequence (SEQ ID NO:6) (14B).
FIG. 15 depicts the generation of domain swap construct by PCR-SOE method. Arrow bars represent PCR primers. Open bar, Notch3 sequence. Filled bar, Notch1 sequence.
FIG. 16 depicts the amino acid sequences used in the Notch3 LIN12 domain epitope mapping of the MAb 256A-4 and 256A-8.
FIG. 17 depicts the amino acid sequences used in the Notch3 dimerization domain epitope mapping of the MAb 256A-4 and 256A-8.
FIG. 18 depicts a schematic of the epitope binding site for MAb 256A-4 and 256A-8.



DETAILED DESCRIPTION
This invention is not limited to the particular methodology, protocols, cell lines, vectors, or reagents described herein because they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise, e.g., reference to “a host cell” includes a plurality of such host cells. Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.
All patents and publications mentioned herein are incorporated herein by reference to the extent allowed by law for the purpose of describing and disclosing the proteins, enzymes, vectors, host cells, and methodologies reported therein that might be used with the present invention. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Definitions
Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definitions as defined below.
The phrase “substantially identical” with respect to an antibody chain polypeptide sequence may be construed as an antibody chain exhibiting at least 70%, or 80%, or 90%, or 95% sequence identity to the reference polypeptide sequence. The term with respect to a nucleic acid sequence may be construed as a sequence of nucleotides exhibiting at least about 85%, or 90%, or 95%, or 97% sequence identity to the reference nucleic acid sequence.
The term “identity” or “homology” shall be construed to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity may be measured using sequence analysis software.
The term “antibody” is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. The antibodies 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. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
As used herein, “anti-Notch3 antibody” means an antibody which binds specifically to human Notch3 in such a manner so as to inhibit or substantially reduce the binding of Notch3 to its ligands or to inhibit Notch 3 signaling.
The term “variable” in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular target. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely a adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target binding site of antibodies (see Kabat, et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. (1987)). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat, et al., unless otherwise indicated.
The term “antibody fragment” refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include F(ab), F(ab′), F(ab′)2 and Fv fragments. The phrase “functional fragment or analog” of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-Notch3 antibody is one which can bind to a Notch3 receptor in such a manner so as to prevent or substantially reduce the ability of the receptor to bind to its ligands or initiate signaling. As used herein, “functional fragment” with respect to antibodies, refers to Fv, F(ab) and F(ab′)2 fragments. An “Fv” fragment consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site.
“Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding.
The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another changing and create two antigen-binding sites.
The F(ab) fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. F(ab′) fragments differ from F(ab) fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. F(ab′) fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab′)2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984)). Monoclonal antibodies are highly specific, being directed against a single target site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the target. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies for use with the present invention may be isolated from phage antibody libraries using well known techniques. The parent monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, et al., Nature 256:495 (1975), or may be made by recombinant methods.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other target-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin template chosen.
The terms “cell,” “cell line,” and “cell culture” include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included. The “host cells” used in the present invention generally are prokaryotic or eukaryotic hosts.
“Transformation” of a cellular organism, cell, or cell line with DNA means introducing DNA into the target cell so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration. “Transfection” of a cell or organism with DNA refers to the taking up of DNA, e.g., an expression vector, by the cell or organism whether or not any coding sequences are in fact expressed. The terms “transfected host cell” and “transformed” refer to a cell in which DNA was introduced. The cell is termed “host cell” and it may be either prokaryotic or eukaryotic. Typical prokaryotic host cells include various strains of E. coli. Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary or cells of human origin. The introduced DNA sequence may be from the same species as the host cell or a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA.
The term “vector” means a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA sequence in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of vectors which serve equivalent function as and which are, or become, known in the art.
“Mammal” for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
The word “label” when used herein refers to a detectable compound or composition which can be conjugated directly or indirectly to a molecule or protein, e.g., an antibody. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
As used herein, “solid phase” means a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column).
As used herein, the term “Notch3-mediated disorder” means a condition or disease which is characterized by the overexpression and/or hypersensitivity of the Notch3 receptor. Specifically it would be construed to include conditions associated with cancers such as non-small cell lung cancer, ovarian cancer, and T-cell acute lymphoblastic leukemia. Other cancers including pancreatic, prostate cancer, plasma cell neoplasms (e.g., multiple myeloma, plasma cell leukemia and extramedullary plasmacytoma), neuroblastoma and extramedullary plasmacytoma are also encompassed under the scope of this term. Other types of diseases include lymphoma, Alagille syndrome, liver disease involving aberrant vascularization, neurologic diseases, diabetes, diseases involving vascular cell fate, and rheumatoid arthritis.
Notch 3 Receptor Immunogen for Generating Antibodies
Soluble targets or fragments thereof can be used as immunogens for generating antibodies. The antibody is directed against the target of interest. Preferably, the target is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal. Whole cells may be used as the immunogen for making antibodies. The immunogen may be produced recombinantly or made using synthetic methods. The immunogen may also be isolated from a natural source.
For transmembrane molecules, such as receptors, fragments of these (e.g., the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells can be derived from a natural source (e.g., cancer cell lines) or may be cells which have been transformed by recombinant techniques to over-express the transmembrane molecule. Other forms of the immunogen useful for preparing antibodies will be apparent to those in the art.
Alternatively, a gene or a cDNA encoding human Notch3 receptor may be cloned into a plasmid or other expression vector and expressed in any of a number of expression systems according to methods well known to those of skill in the art. Methods of cloning and expressing Notch3 receptor and the nucleic acid sequence for human Notch3 receptor are known (see, for example, U.S. Pat. Nos. 5,821,332 and 5,759,546). Because of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding Notch3 receptor protein or polypeptides may be used. One may vary the nucleotide sequence by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence that codes for naturally occurring Notch3 receptor and all such variations may be considered. Any one of these polypeptides may be used in the immunization of an animal to generate antibodies that bind to human Notch3 receptor.
Recombinant Notch3 proteins from other species may also be used as immunogen to generate antibodies because of the high degree of conservation of the amino acid sequence of Notch3. A comparison between human and mouse Notch3 showed over 90% amino acid sequence identity between the two species.
The immunogen Notch3 receptor may, when beneficial, be expressed as a fusion protein that has the Notch3 receptor attached to a fusion segment. The fusion segment often aids in protein purification, e.g., by permitting the fusion protein to be isolated and purified by affinity chromatography, but can also be used to increase immunogenicity. Fusion proteins can be produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of the protein. Fusion segments may include, but are not limited to, immunoglobulin Fc regions, glutathione-S-transferase, β-galactosidase, a poly-histidine segment capable of binding to a divalent metal ion, and maltose binding protein.
Recombinant Notch3 receptor protein as described in Example 1 was used to immunize mice to generate the hybridomas that produce the monoclonal antibodies of the present invention. Exemplary polypeptides comprise all or a portion of SEQ ID NO. 1 or variants thereof.
Antibody Generation
The antibodies of the present invention may be generated by any suitable method known in the art. The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: a Laboratory Manual, Cold spring Harbor Laboratory Press, 2nd ed. (1988), which is hereby incorporated herein by reference in its entirety).
For example, an immunogen as described in Example 1 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. The administration of the immunogen may entail one or more injections of an immunizing agent and, if desired, an adjuvant. 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. Additional examples of adjuvants which may be employed include the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Immunization protocols are well known in the art and may be performed by any method that elicits an immune response in the animal host chosen. Adjuvants are also well known in the art.
Typically, the immunogen (with or without adjuvant) is injected into the mammal by multiple subcutaneous or intraperitoneal injections, or intramuscularly or through IV. The immunogen may include a Notch3 polypeptide, a fusion protein, or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunogen to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivatizing active chemical functional groups to both the immunogen and the immunogenic protein to be conjugated such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, ovalbumin, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor, and promiscuous T helper peptides. Various adjuvants may be used to increase the immunological response as described above.
The antibodies of the present invention comprise monoclonal antibodies. Monoclonal antibodies are antibodies which recognize a single antigenic site. Their uniform specificity makes monoclonal antibodies much more useful than polyclonal antibodies, which usually contain antibodies that recognize a variety of different antigenic sites. Monoclonal antibodies may be prepared using hybridoma technology, such as those described by Kohler, et al., Nature 256:495 (1975); U.S. Pat. No. 4,376,110; Harlow, et al., Antibodies: A Laboratory Manual, Cold spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier (1981), recombinant DNA methods, or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies include, but are not limited to, the human B-cell hybridoma technique (Kosbor, et al., Immunology Today 4:72 (1983); Cole, et al., Proc Natl Acad Sci USA 80:2026 (1983)), and the EBV-hybridoma technique (Cole, et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss (1985)). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo.
In the hybridoma model, a host such as a mouse, a humanized mouse, a mouse with a human immune system, hamster, rabbit, camel, or any other appropriate host animal, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).
Generally, in making antibody-producing hybridomas, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine or human origin. Typically, a rat or mouse myeloma cell line is employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), substances that prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines, such as those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., and SP2/0 or X63-Ag8-653 cells available from the American Type Culture Collection (ATCC), Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J Immunol 133:3001 (1984); Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc, pp. 51-63 (1987)). The mouse myeloma cell line NSO may also be used (European Collection of Cell Cultures, Salisbury, Wilshire, UK).
The culture medium in which hybridoma cells are grown is assayed for production of monoclonal antibodies directed against Notch3. The binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody to Notch3 can, for example, be determined by a Scatchard analysis (MUNSON, et al., Anal Biochem 107:220 (1980)).
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium (D-MEM) or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated or isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-SEPHAROSE® affinity media, hydroxylaptite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.
A variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hybridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources) (Innis, et al. In PCR Protocols. A Guide to Methods and Applications, Academic (1990), Sanger, et al., Proc Natl Acad Sci 74:5463 (1977)). The hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, NS0 cells, Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.
Antibody fragments which recognize specific epitopes may be generated by known techniques. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto, et al., J Biochem Biophys Methods 24:107 (1992); Brennan, et al., Science 229:81 (1985)). 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 CH1 domain of the heavy chain. However, these fragments can now be produced directly by recombinant host ells. For example, the antibody fragments can be isolated from an antibody phage library. Alternatively, F(ab′)2-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter, et al., Bio/Technology 10:163 (1992). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (Fv) (PCT patent application WO 93/16185).
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., J Immunol Methods 125:191 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.
A humanized antibody is designed to have greater homology to a human immunoglobulin than animal-derived monoclonal antibodies. Humanization is a technique for making a chimeric antibody wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. Humanized antibodies are antibody molecules generated in a non-human species that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework (FR) 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., 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 28:489 (1991); Studnicka, et al., Protein Engineering 7:805 (1994); Roguska, et al., Proc Natl Acad Sci USA 91:969 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones, et al., Nature 321:522 (1986); Riechmann, et al., Nature 332:323 (1988); Verhoeyen, et al., Science 239:1534 (1988)), by substituting non-human CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.
It is further important that humanized antibodies retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin sequences, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is maximized, although it is the CDR residues that directly and most substantially influence antigen binding.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is important to reduce antigenicity. According to an exemplary method, the so-called “best-fit” method, the sequence of the variable domain of a non-human antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of that of the non-human parent antibody is then accepted as the human FR for the humanized antibody (Sims, et al., J Immunol 151:2296 (1993); Chothia, et al., J Mol Biol 196:901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter, et al., Proc Natl Acad Sci USA 89:4285 (1992); Presta, et al., J Immunol 151:2623 (1993)).
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. The techniques of Cole, et al. and Boerder, et al. are also available for the preparation of human monoclonal antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss (1985); and Boerner, et al., J Immunol 147:86 (1991)).
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. See, e.g., Jakobovits, et al., Proc Natl Acad Sci USA 90:2551 (1993); Jakobovits, et al., Nature 362:255 (1993); Bruggermann, et al., Year in Immunol 7:33 (1993); Duchosal, et al., Nature 355:258 (1992)). 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 can 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, et al., 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; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Also human mAbs could be made by immunizing mice transplanted with human peripheral blood leukocytes, splenocytes or bone marrows (e.g., Trioma techniques of XTL). 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 (1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art (See, e.g., Greenspan, et al., FASEB J 7:437 (1989); Nissinoff, J Immunol 147:2429 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards Notch3, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.
Methods for making bispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein, et al., Nature 305:537 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829 and in Traunecker, et al., EMBO J 10:3655 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It may have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh, et al., Meth In Enzym 121:210 (1986).
Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
In addition, one can generate single-domain antibodies to Notch3. Examples of this technology have been described in WO94/25591 for antibodies derived from Camelidae heavy chain Ig, as well in US2003/0130496 describing the isolation of single domain fully human antibodies from phage libraries.
One can also create a single peptide chain binding molecules in which the heavy and light chain Fv regions are connected. Single chain antibodies (“scFv”) and the method of their construction are described in U.S. Pat. No. 4,946,778. Alternatively, Fab can be constructed and expressed by similar means. All of the wholly and partially human antibodies are less immunogenic than wholly murine mAbs, and the fragments and single chain antibodies are also less immunogenic.
Antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty, et al., Nature 348:552 (1990). Clarkson, et al., Nature 352:624 (1991) and Marks, et al., J Mol Biol 222:581 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks, et al., Bio/Technology 10:779 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse, et al., Nuc Acids Res 21:2265 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc Natl Acad Sci USA 81:6851 (1984)).
Another alternative is to use electrical fusion rather than chemical fusion to form hybridomas. This technique is well established. Instead of fusion, one can also transform a B cell to make it immortal using, for example, an Epstein Barr Virus, or a transforming gene. See, e.g., “Continuously Proliferating Human Cell Lines Synthesizing Antibody of Predetermined Specificity,” Zurawaki, et al., in Monoclonal Antibodies, ed. by Kennett, et al., Plenum Press, pp. 19-33. (1980)). Anti-Notch3 mAbs can be raised by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with Notch3 protein, fusion protein, or its fragments expressed by either eukaryotic or prokaryotic systems. Other animals can be used for immunization, e.g., non-human primates, transgenic mice expressing immunoglobulins, and severe combined immunodeficient (SCID) mice transplanted with human B lymphocytes. Hybridomas can be generated by conventional procedures by fusing B lymphocytes from the immunized animals with myeloma cells (e.g., Sp2/0 and NSO), as described earlier (Köhler, et al., Nature 256:495 (1975)). In addition, anti-Notch3 antibodies can be generated by screening of recombinant single-chain Fv or Fab libraries from human B lymphocytes in phage-display systems. The specificity of the mAbs to Notch3 can be tested by ELISA, Western immunoblotting, or other immunochemical techniques. The inhibitory activity of the antibodies on complement activation can be assessed by hemolytic assays, using sensitized chicken or sheep RBCs for the classical complement pathway. The hybridomas in the positive wells are cloned by limiting dilution. The antibodies are purified for characterization for specificity to human Notch3 by the assays described above.
Identification of Anti-Notch-3 Antibodies
The present invention provides antagonist monoclonal antibodies that inhibit and neutralize the action of Notch3. In particular, the antibodies of the present invention bind to and inhibit the activation of Notch3. The antibodies of the present invention include the antibodies designated 256A-4 and 256A-8, which are disclosed herein. The present invention also includes antibodies that bind to the same epitope as one of these antibodies.
Candidate anti-Notch3 antibodies were tested by enzyme linked immunosorbent assay (ELISA), Western immunoblotting, or other immunochemical techniques. Assays performed to characterize the individual antibodies are described in the Examples.
Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, single-domain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
The antibodies may be human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and single-domain antibodies 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. Also included in the invention are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are from human, non-human primates, rodents (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.
As used herein, “human” antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati, et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of Notch3 or may be specific for both Notch3 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., J Immunol 147:60 (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 (1992).
Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of Notch3 which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that bind Notch3 polypeptides, which have 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 Notch3 are also included in the present invention. Anti-Notch3 antibodies may also bind with a KD of less than about 10−7 M, less than about 10−6 M, or less than about 10−5 M to other proteins, such as anti-Notch3 antibodies from species other than that against which the anti-Notch3 antibody is directed.
In specific embodiments, antibodies of the present invention cross-react with monkey homologues of human Notch3 and the corresponding epitopes thereof. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of the specific antigenic and/or immunogenic polypeptides disclosed herein.
Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide encoding Notch3 under stringent hybridization conditions. Antibodies of the present 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 an equilibrium dissociation constant or KD from 10−8 to 10−15 M, 10−8 to 10−12 M, 10−8 to 10−10 M, or 10−10 to 10−12 M. The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention 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%.
Vectors and Host Cells
In another aspect, the present invention provides isolated nucleic acid sequences encoding an antibody as disclosed herein, vector constructs comprising a nucleotide sequence encoding the antibodies of the present invention, host cells comprising such a vector, and recombinant techniques for the production of the antibody.
For recombinant production of an antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Standard techniques for cloning and transformation may be used in the preparation of cell lines expressing the antibodies of the present invention.
Vectors
Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Recombinant expression vectors containing a nucleotide sequence encoding the antibodies of the present invention can be prepared using well known techniques. The expression vectors include a nucleotide sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences such as those derived from mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators, enhancers, mRNA ribosomal binding sites, and/or other appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleotide sequence for the appropriate polypeptide. Thus, a promoter nucleotide sequence is operably linked to, e.g., the antibody heavy chain sequence if the promoter nucleotide sequence controls the transcription of the appropriate nucleotide sequence.
In addition, sequences encoding appropriate signal peptides that are not naturally associated with antibody heavy and/or light chain sequences can be incorporated into expression vectors. For example, a nucleotide sequence for a signal peptide (secretory leader) may be fused in-frame to the polypeptide sequence so that the antibody is secreted to the periplasmic space or into the medium. A signal peptide that is functional in the intended host cells enhances extracellular secretion of the appropriate antibody. The signal peptide may be cleaved from the polypeptide upon secretion of antibody from the cell. Examples of such secretory signals are well known and include, e.g., those described in U.S. Pat. Nos. 5,698,435; 5,698,417; and 6,204,023.
The vector may be a plasmid vector, a single or double-stranded phage vector, or a single or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides by well known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also may be introduced into cells as packaged or encapsulated virus by well known techniques for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. Cell-free translation systems may also be employed to produce the protein using RNAs derived from the present DNA constructs. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publications WO 86/05807 and 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.
Host Cells
The antibodies of the present invention can be expressed from any suitable host cell. Examples of host cells useful in the present invention include prokaryotic, yeast, or higher eukaryotic cells and 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).
Prokaryotes useful as host cells in the present invention include gram negative or gram positive organisms such as E. coli, B. subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella, as well as Bacilli, Pseudomonas, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the pKK223-3 vector (Pharmacia Fine Chemicals, Uppsala, Sweden), PGEM® 1 vector (Promega Biotec, Madison, Wis., USA), and the pET (Novagen, Madison, Wis., USA) and pRSET (Invitrogen, Carlsbad, Calif.) series of vectors (Studier, J Mol Biol 219:37 (1991); Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include T7, (Rosenberg, et al., Gene 56:125 (1987)), β-lactamase (penicillinase), lactose promoter system (Chang, et al., Nature 275:615 (1978); Goeddel, et al., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddel, et al., Nucl Acids Res 8:4057 (1980)), and tac promoter (Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990)).
Yeasts or filamentous fungi useful in the present invention include those from the genus Saccharomyces, Pichia, Actinomycetes, Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma, Neurospora, and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman, et al., J Biol Chem 255:2073 (1980)) or other glycolytic enzymes (Holland, et al., Biochem 17:4900 (1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Fleer, et al., Gene 107:285 (1991). Other suitable promoters and vectors for yeast and yeast transformation protocols are well known in the art. Yeast transformation protocols are well known. One such protocol is described by Hinnen, et al., Proc Natl Acad Sci 75:1929 (1978). The Hinnen protocol selects for Trp+ transformants in a selective medium.
Mammalian or insect host cell culture systems may also be employed to express recombinant antibodies. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells (Luckow, et al., Bio/Technology 6:47 (1988); Miller, et al., Genetics Engineering, Setlow, et al., eds. Vol. 8, pp. 277-9, Plenam Publishing (1986); Mseda, et al., Nature 315:592 (1985)). For example, Baculovirus systems may be used for production of heterologous proteins. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) may be 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). Other hosts that have been identified include Aedes, Drosophila melanogaster, and Bombyx mori. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of AcNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Moreover, plant cells cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco and also be utilized as hosts.
Vertebrate cells, and propagation of vertebrate cells, in culture (tissue culture) has become a routine procedure. See Tissue Culture, Kruse, et al., eds., Academic Press (1973). Examples of useful mammalian host cell lines are monkey kidney; human embryonic kidney line; baby hamster kidney cells; Chinese hamster ovary cells/−DHFR (CHO, Urlaub, et al., Proc Natl Acad Sci USA 77:4216 (1980)); mouse sertoli cells; human cervical carcinoma cells (HELA); canine kidney cells; human lung cells; human liver cells; mouse mammary tumor; and NS0 cells.
Host cells are transformed with the above-described vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, transcriptional and translational control sequences, selecting transformants, or amplifying the genes encoding the desired sequences. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, Adenovirus 2, Simian virus 40 (SV40), and human cytomegalovirus (CMV). DNA sequences derived from the SV40 viral genome may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell, e.g., SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication. Exemplary expression vectors for use in mammalian host cells are commercially available.
The host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma, St Louis, Mo.), Minimal Essential Medium (MEM, Sigma, St Louis, Mo.), RPMI-1640 (Sigma, St Louis, Mo.), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma, St Louis, Mo.) are suitable for culturing host cells. In addition, any of the media described in Ham, et al., Meth Enzymol 58:44 (1979), Barnes, et al., Anal Biochem 102:255 (1980), and U.S. Pat. Nos. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as X-chlorides, where X is sodium, calcium, magnesium; and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
Polynucleotides Encoding Antibodies
The invention further provides polynucleotides or nucleic acids, e.g., DNA, comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. Exemplary polynucleotides include those encoding antibody chains comprising one or more of the amino acid sequences described herein. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions to polynucleotides that encode an antibody of the present invention.
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., Bio/Techniques 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, or a 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 of the invention) 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.
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., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1990); Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1998), 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 CDRs by well known methods, 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 (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 (1984); Neuberger, et al., Nature 312:604 (1984); Takeda, et al., Nature 314:452 (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 (1988); Huston, et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al., Nature 334:544 (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 may also be used (Skerra, et al., Science 242:1038 (1988)).
Methods of Producing Anti-Notch3 Antibodies
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment, derivative, or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody or a fragment of the antibody. Once a polynucleotide encoding an antibody molecule has been obtained, the vector for the production of the antibody may be produced by recombinant DNA technology. An expression vector is constructed 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 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 of the invention. In one aspect of the invention, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention as described above. 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. Bacterial cells such as E. coli, and eukaryotic cells are commonly used for the expression of a recombinant antibody molecule, especially for the expression of whole recombinant antibody molecule. For example, mammalian cells such as CHO, in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking, et al., Gene 45:101 (1986); Cockett, et al., Bio/Technology 8:2 (1990)).
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, COS, 293, 3T3, or myeloma cells.
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 one to two 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, et al., 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., Proc 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, et al., Proc Natl Acad Sci USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu, et al., Biotherapy 3:87 (1991)); and hygro, which confers resistance to hygromycin (Santerre, 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 (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press (1990); and in Chapters 12 and 13, Dracopoli, et al., eds, Current Protocols in Human Genetics, John Wiley & Sons (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, et al., “The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells,” DNA Cloning, Vol. 3. Academic Press (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 gene, production of the antibody will also increase (Crouse, et al., Mol Cell Biol 3:257 (1983)).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived 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 size-exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide. Fused or conjugated antibodies of the present invention may be used for ease in purification. See e.g., PCT publication WO 93/21232; EP 439,095; Naramura, et al., Immunol Lett 39:91 (1994); U.S. Pat. No. 5,474,981; Gillies, et al., Proc Natl Acad Sci USA 89:1428 (1992); Fell, et al., J Immunol 146:2446 (1991), which are incorporated by reference in their entireties.
Moreover, the antibodies or fragments thereof of the present invention 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., Valencia, Calif.), among others, many of which are commercially available. As described in Gentz, et al., Proc Natl Acad Sci USA 86:821 (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.
Antibody Purification
When using recombinant techniques, an antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Carter, et al., Bio/Technology 10:163 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human IgG1, IgG2 or IgG4 heavy chains (Lindmark, et al., J Immunol Meth 62:1 (1983)). Protein G is recommended for all mouse isotypes and for human IgG3 (Guss, et al., EMBO J 5:1567 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTM resin (J. T. Baker; Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE® chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
Pharmaceutical Formulation
Therapeutic formulations of the polypeptide or antibody may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired degree of purity with optional “pharmaceutically-acceptable” carriers, excipients or stabilizers typically employed in the art (all of which are termed “excipients”), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See Remington's Pharmaceutical Sciences, 16th edition, Osol, Ed. (1980). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are preferably present at concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, there may be mentioned phosphate buffers, histidine buffers and trimethylamine salts such as Tris.
Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.2%-1% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
Isotonicifiers sometimes known as “stabilizers” may be added to ensure isotonicity of liquid compositions of the present invention and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha.-monothioglycerol and sodium thio sulfate; low molecular weight polypeptides (i.e. <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccacharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in the range from 0.1 to 10,000 weights per part of weight active protein.
Non-ionic surfactants or detergents (also known as “wetting agents”) may be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), PLURONIC® polyols, polyoxyethylene sorbitan monoethers (TWEEN-20®, TWEEN-80®, etc.). Non-ionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
Additional miscellaneous excipients include bulking agents, (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents. The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an immunosuppressive agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in microcapsule prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osal, Ed. (1980).
The formulations to be used for in vivo administration should be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C. resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
The amount of therapeutic polypeptide, antibody, or fragment thereof which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the dose-response curve and the pharmaceutical compositions of the invention first in vitro, and then in useful animal model systems prior to testing in humans.
In a preferred embodiment, an aqueous solution of therapeutic polypeptide, antibody or fragment thereof is administered by subcutaneous injection. Each dose may range from about 0.5 μg to about 50 μg per kilogram of body weight, or more preferably, from about 3 μg to about 30 μg per kilogram body weight.
The dosing schedule for subcutaneous administration may vary from once a month to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the subject's sensitivity to the therapeutic agent.
Therapeutic Uses of Anti-Notch-3 Antibodies
It is contemplated that the antibodies of the present invention may be used to treat a mammal. In one embodiment, the antibody is administered to a nonhuman mammal for the purposes of obtaining preclinical data, for example. Exemplary nonhuman mammals to be treated include nonhuman primates, dogs, cats, rodents and other mammals in which preclinical studies are performed. Such mammals may be established animal models for a disease to be treated with the antibody or may be used to study toxicity of the antibody of interest. In each of these embodiments, dose escalation studies may be performed on the mammal.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) can be used as a therapeutic. The present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, a mammal, or a human, for treating a Notch3-mediated disease, disorder, or condition. The animal or subject may be a mammal in need of a particular treatment, such as a mammal having been diagnosed with a particular disorder, e.g., one relating to Notch3. Antibodies directed against Notch3 are useful against cancer and other Notch3-associated diseases including neurological disorders, diabetes, rheumatoid arthritis, vascular related diseases, and Alagille syndrome in mammals, including but not limited to cows, pigs, horses, chickens, cats, dogs, non-human primates etc., as well as humans. For example, by administering a therapeutically acceptable dose of an anti-Notch3 antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, disease symptoms may be ameliorated or prevented in the treated mammal, particularly humans.
Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention as described below (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 Notch3, 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 Notch3 includes, but is not limited to, alleviating at least one symptom associated with those diseases, disorders, or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
Anti-Notch3 antibodies of the present invention may be used therapeutically in a variety of diseases. The present invention provides a method for preventing or treating Notch3-mediated diseases in a mammal. The method comprises administering a disease preventing or treating amount of anti-Notch3 antibody to the mammal. The anti-Notch3 antibody binds to Notch3 and antagonizes its function. Notch3 signaling has been linked to various diseases such as various cancers (Haruki, et al., Cancer Res 65:3555 (2005); Park, et al., Cancer Res 66:6312 (2006); Lu, et al., Clin Cancer Res 10:3291 (2004)); Hedvat, et al., Br J Haematol 122:728 (2003); Buchler, et al., Ann Surg 242:791 (2005)); Bellavia, et al., Proc Natl Acad Sci USA 99:3788 (2002); Screpanti, et al., Trends Mol Med 9:30 (2003)); van Limpt, et al., Cancer Lett 228:59 (2005)), neurological disorders (Joutel, et al., Nature 383:707 (1996)), diabetes (Anastasi, et al., J Immunol 171:4504 (2003), rheumatoid arthritis (Yabe, et al., J Orthop Sci 10:589 (2005)), vascular related diseases (Sweeney, et al., FASEB J 18:1421 (2004)), and Alagille syndrome (Flynn, et al., J Pathol 204:55 (2004)). Anti-Notch3 antibodies will also be effective to prevent the above mentioned diseases.
The amount of the antibody which will be effective in the treatment, inhibition, and prevention of a disease or disorder associated with aberrant expression and/or activity of Notch3 can be determined by standard clinical techniques. The dosage will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody can be administered in treatment regimes consistent with the disease, e.g., a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence. 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 150 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. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
The antibody composition will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “therapeutically effective amount” of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat a disease or disorder. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
The antibodies of the invention may be administered alone or in combination with other types of cancer treatments including conventional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin and doxorbicin), anti-EGFR agents (gefitinib, erlotinib and cetuximab), anti-angiogenesis agents (bevacizumab and sunitinib), as well as immuno-modulating agents such as interferon-α and thalidomide.
In a preferred aspect, the antibody is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
Various delivery systems are known and can be used to administer an antibody of the present invention, including injection, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu, et al., J Biol Chem 262:4429 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
The anti-Notch3 antibody can be administered to the mammal in any acceptable manner. Methods of introduction include but are not limited to parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation, and oral routes, and if desired for immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial, or intraperitoneal administration. The antibodies 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 therapeutic antibodies 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. In addition, the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. The antibody may also be administered into the lungs of a patient in the form of a dry powder composition (See e.g., U.S. Pat. No. 6,514,496).
In a specific embodiment, it may be desirable to administer the therapeutic antibodies 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, topical application, 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 an antibody of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the antibody can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527 (1990); Treat, et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein, et al., eds., pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-27; see generally ibid.).
In yet another embodiment, the antibody can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, Science 249:1527 (1990); 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, et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen, et al., eds., Wiley (1984); Ranger, et al., 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.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the antibody and a physiologically acceptable carrier. In a specific embodiment, the term “physiologically 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 physiological 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 carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain an effective amount of the antibody, 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 one 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 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 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.
In addition, the antibodies of the present invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. An antibody or fragment thereof 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 homologues 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-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).
Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon, et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies and Cancer Therapy, Reisfeld, et al. (eds.), pp. 243-56 Alan R. Liss (1985); Hellstrom, et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery, 2nd ed., Robinson, et al., eds., pp. 623-53, Marcel Dekker (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., Immunol Rev 62:119 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate. See, e.g., U.S. Pat. No. 4,676,980.
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, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-α, TNF-β, 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 (1994)), VEGI (See, International Publication No. WO 99/23105); a thrombotic agent; 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.
Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for preventing or treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is the antibody. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
Antibody-Based Gene Therapy
In a another aspect of the invention, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of Notch3, 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 can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel, et al., Clinical Pharmacy 12:488 (1993); Wu, et al., Biotherapy 3:87 (1991); Tolstoshev, Ann Rev Pharmacol Toxicol 32:573 (1993); Mulligan, Science 260:926 (1993); Morgan, et al., Ann Rev Biochem 62:191 (1993); May, TIBTECH 11:155 (1993).
In a one aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific.
In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller, et al., Proc Natl Acad Sci USA 86:8932 (1989); Zijlstra, et al., Nature 342:435 (1989)). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu, et al., J Biol Chem 262:4429 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller, et al., Proc Natl Acad Sci USA 86:8932 (1989); Zijlstra, et al., Nature 342:435 (1989)).
In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller, et al., Meth Enzymol 217:581 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitate the delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen, et al., Biotherapy 6:291 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes, et al., J Clin Invest 93:644 (1994); Kiem, et al., Blood 83:1467 (1994); Salmons, et al., Human Gene Therapy 4:129 (1993); and Grossman, et al., Curr Opin Gen and Dev 3:110 (1993).
Adenoviruses may also be used in the present invention. Adenoviruses are especially attractive vehicles in the present invention for delivering antibodies to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky, et al., Curr Opin Gen Dev 3:499 (1993) present a review of adenovirus-based gene therapy. Bout, et al., Human Gene Therapy 5:3 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld, et al., Science 252:431 (1991); Rosenfeld, et al., Cell 68:143 (1992); Mastrangeli, et al., J Clin Invest 91:225 (1993); PCT Publication WO94/12649; Wang, et al., Gene Therapy 2:775 (1995). Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh, et al., Proc Soc Exp Biol Med 204:289 (1993); U.S. Pat. Nos. 5,436,146; 6,632,670; and 6,642,051).
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler, et al., Meth Enzymol 217:599 (1993); Cohen, et al., Meth Enzymol 217:618 (1993); Cline, Pharmac Ther 29:69 (1985)) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In one embodiment, the cell used for gene therapy is autologous to the patient. Nucleic acid sequences encoding an antibody of the present invention are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple, et al., Cell 71:973 (1992); Rheinwald, Meth Cell Bio 21A:229 (1980); Pittelkow, et al., Mayo Clinic Proc 61:771 (1986)).
EXAMPLES
Example 1
Generation of Immunogen: Notch3 Extracellular Domain-Fc Fusion Protein
Anti-Notch3 monoclonal antibodies that specifically bind to the LIN12/dimerization domain (herein after “LD”) of human Notch3 were generated using a recombinant Notch3-Fc fusion protein as immunogen comprising Notch3 LD fused to a gamma 1 Fc region at the carboxy terminal end. Specifically, the immunogen comprised amino acid residues 1378 to 1640 of Notch3 LD (See FIG. 1) and human γ1Fc fusion protein (Notch3 LD/Fc). A control antibody was generated comprising the Notch3 EGF repeat region from amino acid residues 43 to 1377 (designated 255A-79).
Notch3 protein sequence was analyzed using an internet-based research software and service (Motif Search). Human liver and pancreatic RNAs (Ambion, Inc. Austin, Tex.) were used as templates to synthesize the first strand of cDNA using a standard commercially available cDNA synthesis kit. The cDNAs encoding the Notch3 LD and the EGF repeat region were PCR-amplified in the presence of Betaine (1-2M) and DMSO (5%). The PCR-synthesized Notch3-LD DNA fragment ( ̃0.8 kb) and Notch3-EGF repeat DNA fragment ( ̃4 kb) were cloned into expression vectors comprising a Hisγ1Fc in the commercially available vector pSec or in the commercially available vector pCD3.1, each bearing a different antibiotic marker. This cloning resulted in two expression plasmids, one expressing a Notch3-LD/Fc fusion protein and the other expressing a Notch3-EGF/Fc fusion protein.
To facilitate the plasmid construction and to enhance the expression of the various Notch 3 recombinant proteins, oligonucleotides corresponding to the leader peptide sequence comprising the first 135 base pairs of the Notch3 nucleic acid coding sequence were generated. These oligonucleotides contained some changes in the wobble coding positions to lower the GC content. All nucleotide sequence changes were silent, i.e., no amino acid sequence changes (FIG. 14A). After annealing the oligonucleotides together, the engineered leader peptide coding sequence was linked to the rest of the coding sequence by PCR-SOE (Ho, et al., Gene 77:51 (1989); Horton, et al., Bio Techniques 8:528 (1990)) (See FIG. 15). This leader peptide coding sequence was used in Notch3-LD/Fc and Notch3 expression constructs. Therefore, both of the Fc fusion proteins comprise a signal peptide linked to the N-terminus, and a human γ1Fc sequence fused to the C-terminus. The amino acid sequence of Notch3-LD, including the leader peptide, is shown in FIG. 14 and SEQ ID NO:6.
Expression of Notch3-EGF/Fc and Notch3-LD/Fc fusion proteins was verified by transient transfection of the Notch3 expression plasmids into 293T (ATCC Number CRL-11268, Manassas, Va.) and CHO cells (Invitrogen, Carlsbad, Calif.), respectively. Prior to transfection, cells were cultured in DMEM (Invitrogen, Carlsbad, Calif.) growth medium containing 10% fetal calf serum (FCS), 2 mM of glutamine, and 1× essential amino acid solution followed by seeding about 3-5×105 cells per well in 6-well plate and growing for approximately 24 hours. Three micrograms each of the Notch3 fusion protein expression plasmids were transfected into cells in each well using a LIPOFECTAMINETM 2000 transfection system (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol. After transfection, the cells were cultured in fresh growth medium and cultured in a CO2 incubator for approximately 40-48 hours before subjecting to Notch3 fusion protein expression analysis. Alternatively, after transfection, the cells were cultured in growth medium for 3-4 hours, then switched to DMEM medium containing 2% FCS and cultured for approximately 60-66 hours before drawing conditioned medium for secreted protein analysis.
Stable cell lines were generated for both Notch3-LD/Fc (His-Fcγ/pSec vector) and Notch3-EGF/Fc (His-Fcγ/pSec vector). Each plasmid was transfected into CHO cells. After transfection, the cells were cultured in DMEM growth medium overnight, then switched to growth medium with 800 μg/ml hygromycin and cultured at least two weeks until the cells not carrying Notch3 expression plasmid were eliminated by the antibiotics. Conditioned media from the stable cell lines were subjected to Western blot analysis.
Stable or transient transfected cells were assayed for expression and secretion of Notch3-LD/Fc or Notch3-EGF/Fc fusion protein. Transfected cells harvested from culture dishes were washed once with phosphate buffered saline (PBS) and resuspended in deionized water, mixed with an equal volume of 2× protein sample loading buffer (BioRad, Hercules, Calif.) and then heated at about 100° C. for 10 minutes. Secreted protein was analyzed using conditioned medium mixed with an equal volume of 2× protein sample loading buffer and heated at 100° C. for 10 minutes. The samples were separated using 4-15% gradient SDS-PAGE. The proteins were transferred from the gel to a PVDF membrane (BioRad, Hercules, Calif.), which was blocked in 5% non-fat dry milk in PBST (PBS with 0.05% TWEEN-20®) for at least one hour prior to transfer of protein.
Notch3-EGF/Fc and Notch3-LD/Fc fusion proteins were detected by incubating with γFc-specific, HRP-conjugated antibody (Sigma, St Louis, Mo.) in blocking buffer for one hour at room temperature. The membrane was washed three times in PBST and developed with a chemiluminescent substrate.
For Notch3 domain/Fc fusion protein purification, CHO stable cell lines as described above were cultured in DMEM with 2% FCS for up to 5 days. One liter of conditioned medium was collected and subjected to protein-A bead-packed column chromatography for affinity binding. The column was washed with PBS, and the bound proteins were eluted in 50 mM citrate buffer (pH 2.8), and the pH was brought to neutral by adding 1 M Tris-HCl buffer (pH 8). Purity of the protein was assessed by protein gel analysis using 4-15% gradient SDS-PAGE. Protein concentration was assayed using Coomassie blue reagent following the manufacturer's protocol (Pierce, Rockford, Ill.). Through this procedure, milligram quantities of Notch3-LD/Fc and Notch3-EGF/Fc protein were purified for immunization and ELISA binding assays.
Example 2
Generation of Anti-Notch3 MAbs
Male A/J mice (Harlan, Houston, Tex.), 8-12 weeks old, were injected subcutaneously with 25 μg of Notch3-EGF/Fc or Notch3-LD/Fc in complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.) in 200 μl of PBS. Two weeks after the injections and three days prior to sacrifice, the mice were again injected intraperitoneally with 25 μg of the same antigen in PBS. For each fusion, single cell suspensions were prepared from spleen of an immunized mouse and used for fusion with Sp2/0 myeloma cells; 5×108 of Sp2/0 and 5×108 of spleen cells were fused in a medium containing 50% polyethylene glycol (M.W. 1450) (Kodak, Rochester, N.Y.) and 5% dimethylsulfoxide (Sigma, St. Louis, Mo.). The cells were then adjusted to a concentration of 1.5×105 spleen cells per 200 μl of the suspension in Iscove medium (Invitrogen, Carlsbad, Calif.), supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, 100 μg/ml of streptomycin, 0.1 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. Two hundred microliters of the cell suspension were added to each well of about sixty 96-well plates. After around ten days, culture supernatants were withdrawn for screening their antibody-binding activity using ELISA.
The 96-well flat bottom IMMULON® II microtest plates (Dynatech Laboratories, Chantilly, Va.) were coated using 100 μl of Notch3-EGF/Fc or Notch3-LD/Fc (0.1 μg/ml) in (PBS) containing 1× Phenol Red and 3-4 drops pHix/liter (Pierce, Rockford, Ill.) and incubated overnight at room temperature. After the coating solution was removed by flicking of the plate, 200 μl of blocking buffer containing 2% BSA in PBST containing 0.1% merthiolate was added to each well for one hour to block non-specific binding. The wells were then washed with PBST. Fifty microliters of culture supernatant from each fusion well were collected and mixed with 50 μl of blocking buffer and then added to the individual wells of the microtiter plates. After one hour of incubation, the wells were washed with PBST. The bound murine antibodies were then detected by reaction with horseradish peroxidase (HRP)-conjugated, Fc-specific goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.). HRP substrate solution containing 0.1% 3,3,5,5-tetramethyl benzidine and 0.0003% hydrogen peroxide was added to the wells for color development for 30 minutes. The reaction was terminated by the addition of 50 ml of 2 M H2SO4/well. The OD at 450 nm was read with an ELISA plate reader (Molecular Devices, Sunnyvale, Calif.).
Among 185 hybridomas isolated and analyzed, two hybridoma clones from mice immunized with Notch3-LD/Fc generated Notch3 antagonizing antibodies, which were further characterized. The ELISA using supernatant from the two hybridoma clones producing MAbs 256A-4 and 256A-8 showed strong binding activity to the purified Notch3 LD/FC fusion protein to which it was generated and did not bind to human Notch1-LD/Fc (LIN/dimerization domain fused to Fc region at the carboxyl terminus) or a control human Fc protein (data not shown) (Table 1). Later studies using functional assays also demonstrated that MAbs 256A-4 and 256A-8 specifically antagonize Notch3 relative to Notch1 and Notch2 (data not shown).







TABLE 1







ELISA OD readings of anti-Notch3


Mabs using hybridoma supernatant












Notch3-LD/Fc

Notch1-LD/Fc













Mean
S.D.
Mean
S.D.

















256A-4
4.000
0.000
0.106
0.004



256A-8
4.000
0.000
0.115
0.014



Control IgG1*
0.064
0.006
0.066
0.006







*Control IgG was an irrelevant IgG1 monoclonal antibody.






The positive hybridoma clones from this primary ELISA screening were further isolated by single colony-picking and a second ELISA assay as described above was done to verify specific binding to the chosen immunogen. The confirmed hybridoma clones were expanded in larger scale cultures. The monoclonal antibodies (MAbs) were purified from the medium of these large scale cultures using a protein A affinity column. The anti-Notch3 MAbs were then characterized using cell-based binding assays, microscopy, Western blot, and FACS analysis.
Example 3
Cell-Based Binding Assays for Anti-Notch3 MAbs
The cell-based binding assays used to characterize the anti-Notch3 MAbs required cloning a full-length human Notch3 open reading frame into a vector, in this case PCDNATM 3.1/Hygro (Invitrogen, Carlsbad, Calif.). The Notch3-coding region was synthesized by RT-PCR using human liver tumor RNA (Ambion, Inc., Austin, Tex.) as a template. The final plasmid construct, Notch3/Hygro, expressed a full-length Notch3 protein as depicted in FIG. 1. A stable cell line expressing Notch3 was generated by transfection of Notch3/Hygro plasmid construct into 293T cells (ATCC No. CRL-11268) using a LIPOFECTAMINETM 2000 kit following the same procedure as described in Example 1. After transfection, the cells were cultured in DMEM growth medium overnight, then reseeded in growth medium with 200 μg/ml hygromycin and cultured for 12-14 days. Well-isolated single colonies were picked and grown in separate wells until enough clonal cells were amplified. Stable 293T clones that were resistant to hygromycin selection and expressed high levels of Notch3 protein were identified by Western blot analysis, and by fluorescent electromicroscopy using polyclonal anti-Notch3 antibodies (R&D Systems, Minneapolis, Minn.).
A partial Notch3 expression plasmid containing only the Notch LIN12/dimerization (LD) domain and the transmembrane (TM) domain was also constructed by PCR and subcloning into PCDNATM 3.1 vector (Invitrogen, Carlsbad, Calif.). This plasmid construct also contains a V5 tag at its C-terminus and was termed Notch3-LDTM/V5. A stable cell line expressing this plasmid, Notch3-LDTM/V5, was generated according to the procedure described in Example 1.
Human Sup-T1 cell line (ATCC No. CRL-1942) naturally expressing Notch3 was also confirmed by Western blot. Sup-T1 cells were grown in RPMI1640 media containing 10% fetal calf serum, 2 mM of glutamine and 1× essential amino acid solution.
Cell-based antibody-binding was assessed using FMATTM (fluorescence macro-confocal high-throughput screening) 8100 HTS System (Applied Biosystems, Foster City, Calif.) following the protocol provided by the manufacturer. Cell lines naturally expressing Notch3 or stably transfected with Notch3 expression constructs were seeded in 96-well plates. Alternatively, transiently transfected 293T or CHO cells were seeded in the 96-well plate. The cells were seeded at a density of 30,000-50,000 cells per well. After 20-24 hours, anti-Notch3 MAbs and 1×PBS reaction buffer were added to the wells and incubated for one hour at 37° C. Cy-5-conjugated anti-mouse IgG antibody was added in the wells after removal of primary antibodies.
Cell-based antibody-binding was also assessed by fluorescence-activated cell sorter (FACS) using an internally generated 293T/Notch3-stable cell line and two cancer lines, human Sup-T1 and A2780 cell lines (UK ECACC No. Cat. No. 93112519), which both naturally express Notch3 (data not shown). Cells were first incubated with anti-Notch3 MAbs in 1×PBS. After three washes, the cells were incubated with fluorescent molecule-conjugated secondary antibody. The cells were resuspended, fixed in 1×PBS with 0.1% paraformaldehyde, and analyzed by FACS (BD Sciences, Palo Alto, Calif.). The results indicated that both MAbs bind to Notch3 receptor expressed either from recombinant plasmid constructs or as native protein in cultured cells (Table 2). However, Western blot showed that when the Notch3 receptor or the Notch3-LD/Fc fusion protein are denatured in SDS-PAGE and transferred to nylon blot membrane, the anti-Notch3 MAbs no longer bind, suggesting a conformational epitope. Transiently transfected 293T cells containing a Notch3/Hygro plasmid were also stained with immunofluorescence as described above and observed by fluorescent microscopy.







TABLE 2







Binding activity of anti-Notch3 MAbs in cell-based


FACS analysis shown as mean fluorescent intensity









Monoclonal Antibody
293T/Notch3-stable cell line
Sup-T1












256A-4
195
43


256A-8
189
45


negative control*
21
23


positive control**
198
74









The cell-based FMATTM and FACS analyses confirmed that both MAbs 256A-4 and 256A-8 indeed bind to the Notch3 receptor expressed either from recombinant plasmid constructs or as native protein in cultured cells (Table 2 and Table 3).







TABLE 3







Summary of anti-Notch3 MAbs binding activity


in cell-based FMAT TM











mAb 256A-4
mAb 256A-8
mAb G3














Notch3 (full-length)/
+
+
−


293T









G3 is a negative control human IgG1 Mab. A positive binding signal was determined based on the FMATTM signal read-out that was significantly higher than G3 and other negative hybridoma clones (p>0.01). The negative signal of G3 FMATTM binding read-out was considered background. Transiently transfected 293T cells with Notch3/Hygro plasmid were also stained with immunofluorescence as described above and observed by fluorescent microscopy.
Example 4
Western Blot Analysis of Anti-Notch3 MAb Binding Activity
Western blot was performed to assess the anti-Notch3 MAbs' binding activity to Notch3 under denaturing conditions, as well as expression levels of Notch3 and other Notch-related proteins in human cell lines. Purified Notch3-LD/Fc fusion protein was combined with protein loading buffer. Protein samples were also prepared from the transiently or stably transfected cells described in Example 1, which were harvested from culture dishes, washed once with PBS, resuspended in total cellular protein extract buffer (Pierce, Rockford, Ill.), and heated at 100° C. for 10 minutes after adding equal volume of 2× protein sample loading buffer. All samples were separated by electrophoresis in a 4-15% gradient SDS-PAGE. The proteins were transferred from gel to PVDF membrane and anti-Notch3 MAbs were applied to the Western blot membrane as the primary detection antibody. An HRP-conjugated secondary antibody was used for detection and the signal generated using a chemiluminescent substrate as described above. Positive control antibodies against human Fc, V5 tag, Notch3 and Notch1 were purchased from Invitrogen, R&D Systems, Santa Cruz Biotechnologies, and Orbigen.
Western blot analysis showed that MAbs 256A-4 and 256A-8 do not bind to Notch3-LD/Fc under denaturing conditions, which is in distinct contrast to the results observed in ELISA and FACS analyses where Notch3 LIN12/heterodimerization domains are maintained in native molecular conformation. Therefore, it is concluded that MAbs 256A-4 and 256A-8 bind to multiple epitopes in Notch3-LD that have to be maintained in their native conformation. This conclusion was confirmed by the results from epitope mapping discussed in Example 8 below.
Example 5
Assessing Functionality of Anti-Notch3 MAbs by Luciferase Reporter Assay
A. Plasmid Constructs
The full length Notch3 expression construct described in Example 3 above was confirmed by sequencing, and is identical to the published sequence depicted in FIG. 1. Human Jagged1 plasmid was obtained from OriGene (Rockville, Md.), and verified by sequencing as identical to NM—000214 (NCBI/GENBANK® accession number). Because the OriGene Jagged1 plasmid did not have an antibiotic selection marker, the Not I fragment containing Jagged1 coding sequence was transferred into PCDNATM 3.1/Hygromycin. A 3.7 Kb subclone of human Jagged2 cDNA was generated by first strand cDNA synthesis from human T-cell leukemia cell line, HH (ATCC No. CRL-2105) and PCR-amplified. The Jagged2 cDNA was subsequently subcloned. The expression of Notch3, Jagged1, and Jagged2 was verified by transient transfection and Western blot as described in Example 4.
To generate a luciferase reporter plasmid for Notch signaling, two complementary oligonucleotide primers containing tandem repeats of CBF1 binding motif were synthesized having the following sequences:







(SEQ ID NO 7)


5′GCTCGAGCTCGTGGGAAAATACCGTGGGAAAATGAACCGTGGGAAAA


 


TCTCGTGG 


(SEQ ID NO 8)


5′GCTCGAGATTTTCCCACGAGATTTTCCCACGGTTC






These two oligoprimers were annealed at 65° C. in 100 mM of NaCl with each oligo at a concentration of 4 mM. After annealing to each other, the primers were extended by PCR. The PCR product was cloned into a commercially available vector. The insert was verified by sequencing, which contains four tandem repeats of CBF1 binding motif and two flanking Xho I sites. The insert was excised using Xho I and ligated downstream of the firefly luciferase reporter coding sequence. After luciferase reporter assay and sequencing analysis, plasmid clones with eight repeats of CBF1 binding motifs were selected and designated CBF1-Luc.
B. Stable Cell Line Generation
Two stable cell lines were generated for functional assays using human embryonic kidney cell lines (HEK293). One cell line contained the Notch3-expressing plasmid and CBF1-Luc reporter plasmid integrated into the nuclear genome. This cell line was generated by cotransfecting Notch3/hygromycin and CBF1-Luc plasmids into 293T cells using LIPOFECTAMINETM 2000 transfection system according to the manufacturer's protocol. Stable transfection cell clones were selected against 200 μg/ml hygromycin in DMEM growth medium, and screened by luciferase reporter assay and Western blot. A cell line with relatively high level of Notch3 expression (based on Western blot) and luciferase activity was selected for use in functional assay, and designated NC85.
The second stable cell line contained a Notch ligand expression construct, such as Jagged1 or Jagged2, or PCDNATM 3.1 as negative control. Stable cell lines expressing human Jagged1 or harboring PCDNATM 3.1 were generated by transfection into 293T cells and selection against hygromycin as described above. Jagged2 was subcloned, transfected into a 293T cell line and expected to be integrated into a specific locus in the genome. Hygromycin-resistant cells were selected as above.
C. Luciferase Reporter Assay Under Coculture Conditions
NC85 cells were mixed and cocultured with another 293T cell line stably expressing human Jagged1 (Jagged1/293T), Jagged2/293F, or PCDNATM 3.1/293T, respectively, for 24 to 48 hours. At the end of the co-culture, the media was removed by aspiration, cells were lysed in 1× Passive Lysis Buffer (E1501, Promega, Madison, Wis.) and luciferase activities were assayed using the Luciferase Assay System following manufacturer's protocol (E1501, Promega, Madison, Wis.) in TD-20/20 luminometer (Turner Designs Instrument, Sunnyvale, Calif.). As illustrated in FIG. 6 and FIG. 7, when NC85 cells were cocultured with Jagged1/293T or with Jagged2/293F, the luciferase activity was increased 2-4 fold as compared to that of coculturing with PCDNATM 3.1/293T cells. To assess the inhibitory effect of anti-Notch3 MAbs, the antibodies were added to the cell culture at beginning of seeding and mixing of cocultured cells. (256-A, 256A-8 and an EGF-Repeat Domain control 255A-79).
D. Luciferase Reporter Assay by Culturing Cells on Notch Ligand-Coated Plates
Regular 96-well tissue culture plates from Becton Dickinson Labware (#18779, Palo Alto, Calif.) were coated with rat Jagged1/Fc, human DLL-4 (R&D Systems, Minneapolis, Minn.) or Human Fc (Jackson ImmunoResearch, West Grove, Pa.), bovine serum albumin (Sigma, St Louis, Mo.). One hundred microliters of each protein (3 μg/ml in PBS) was distributed in a well and maintained at room temperature or 4° C. for at least 8 hours until the coating solution was removed before use. NC85 cells or cancer cells were seeded at 3-5×104 cells per well and allowed to grow for 28-48 hours. The luciferase reporter assay and antibody inhibition assay were performed as described in Section C above. The luciferase reporter assay demonstrated the two MAbs 256A-4 and 256A-8 binding to LIN12/dimerization domain almost completely blocked Jagged1 and Jagged2-induced luciferase reporter activity (FIGS. 6 and 7). In contrast, a MAb specifically binding to Notch3-EGF domain (255A-79), as a control, only inhibited Jagged1-induced luciferase reporter activity (about 60% inhibition, FIG. 6), but not Jagged2-induced luciferase reporter activity (FIG. 7). The ability of MAbs 256A-4 and 256A-8 to block DLL-4-induced luciferase reporter activity is shown in FIG. 8.
Additional functional assays demonstrated that MAbs 256A-4 and 256A-8 inhibited ligand-induced up-regulation of Notch target genes. 293T cells expressing recombinant Notch3 were cultured on Jagged-1-coated plates. In the presence of MAbs 256A-4 and 256A-8, up-regulation of HES5 and HEY2, two Notch target genes, was inhibited, as measured by quantitative RT-PCR (data not shown).
To verify whether the anti-Notch3 MAbs can bind to native Notch3 expressed in human cancer cells and block the receptor signaling, a reporter assay was performed using two ovarian cancer cell lines, OV/CAR3 and A2780. Both 256A-4 and 256A-8 significantly blocked Jagged1-induced Notch signaling mediated by native Notch3 in OV/CAR3 cells (FIG. 9a). Similarly, both MAbs inhibited about 50% of luciferase activity induced by D114 coated on the plate (FIG. 9b). The latter result is consistent with the fact that both Notch1 and Notch3 are expressed in A2780 cells. These results suggest that the anti-Notch3 MAbs can inhibit native Notch3-mediated signaling in cancer cells.
Example 6
Apoptosis Assay
Annexin V is an early apoptotic marker on the cell surface, and the apoptotic cell population can be marked by fluorophore-labeled anti-Annexin V antibody and quantified by FACS analysis. NC85 cells were seeded at 5-6×104 cells per well in Fc- or Jagged1/Fc-coated 96-well plate as described above and maintained in serum-free DMEM medium for 24 hours. Apoptotic cells were stained by FITC-labeled anti-Annexin V antibody (BD Biosciences, Palo Alto, Calif.) and analyzed by FACS. Cells cultured on Jagged1/Fc-coated surface had significantly lower apoptotic cell population comparing to those cultured on Fc-coated plate (FIG. 10). To study the antibody's functional effect, anti-Notch3 MAbs were added in cell culture at the beginning of the study. As shown in FIG. 10, anti-Notch3 MAbs 256A-4 and 256A-8 blocked about 50-65% of the cell survival effect induced by Jagged1.
Example 7
Cell Migration Assays, Invasion Assays, and Morphology Assays
In vitro cell migration and invasion assays are frequently used to assess metastasis potential of cancer cells. These assays were performed to assay the inhibitory effect exerted by the anti-Notch3 MAbs on the tumorgenic 293T/Notch3-stable cell line (NC85). The invasion assay was performed using COSTAR® 48-well insert plate (Sigma-Aldrich, St. Louis, Mo.). The insert divides the well into upper and lower chambers which are separated by a porous membrane (pore diameter=8 μm) at the bottom of the insert. Notch ligands, Jagged1/Fc, DLL-4, or human Fc, were immobilized on the membrane surface as describe in above sections. NC85 cells were seeded at 100,000 cells per well and maintained in serum-free DMEM in the upper chamber and 10% FCS/DMEM in the lower chamber. After 10-24 hours, cells that remained on the top surface of the insert membrane were removed, and the cells that passed the membrane adhering on the bottom of the insert membrane were stained by 0.05% crystal velvet in PBS. The dye was extracted from the cells by 30% acetic acid and absorption readings at 590 nm were recorded. The anti-Notch3 MAbs were added to cell culture 24 hours before seeding NC85 cells in the COSTAR® assay plate and all MAbs were added to the cell culture 24 hours before seeding NC85 cells in the COSTAR® assay plate. Fresh MAbs were added to maintain the same concentration in the migration assay plate. Experimental results are shown in FIG. 11A.
The invasion assay was performed using Becton Dickinson 48-well MATRIGELTM plate (BD Labware, Palo Alto, Calif.). The cell culture well was divided by an insert well into upper and lower chambers, which are separated by a porous membrane (pore diameter=8 μm) at the bottom of the insert well. An optimized density of MATRIGELTM matrix was coated on the membrane top surface and fibronectin was coated on the membrane bottom surface by the manufacturer. NC85, Jagged1/293T, and pcDNA3.1/293T cells were mixed pair-wise such as indicated in FIG. 11B. A total of 6-10×104 cells were seeded in each well in the 48-well MATRIGELTM plate and cultured in growth medium for 24 hours. The cells that remained on top of the insert membrane in the upper chamber were removed and the cells that passed the membrane adhering on the bottom of the insert membrane were stained by 0.05% crystal velvet in PBS. The dye was extracted and absorption measurements were as described in the previous section. MAbs were added at the beginning of the mixed cell culture. The results are shown in FIG. 11B.
The cell migration assay results showed that when NC85 cells were cultured on Jagged1-coated membrane, the activation of Notch3 signaling significantly increased cell migration, and MAbs 256A-4 and 256A-8 clearly inhibited the migration (FIG. 11A). The invasion experiment showed a similar trend (FIG. 11B).
Additionally, the effect of MAbs 256A-4 and 256A-8 on Jagged-1-induced formation of cell “spheres” was examined. When 293T cells over-expressing Notch3 were cultured on Jagged-1-coated plates, the cells formed loosely attached “cell balls” or “spheres.” In the presence of MAbs 256A-4 and 256A-8, however, formation of these cell spheres was inhibited (data not shown).
Example 8
Mapping the Binding Epitope of Anti-Notch3 MAbs
A. Domain Swap Strategy and Rationale
First, the antagonist Notch3 MAbs bind to Notch3 LIN12/dimerization domain (LD), but not to the homologous human Notch1 LIN12/dimerization domain (See FIGS. 12 and 13). Second, the anti-Notch3 MAbs do not bind to denatured Notch3 protein in Western blot as discussed in Example 4, indicating the MAbs bind to conformational epitopes. Third, Notch3 and Notch1 share approximately 55% amino acid sequence homology in the LIN12/dimerization domain, and therefore it was concluded that a domain swap between Notch3 and Notch1 within this region would not disrupt the protein conformation.
B. Generating Domain Swap Fusion Protein Constructs
Sequence analysis indicated that Notch3 has three LIN12 repeats and its dimerization domain is divided into two segments. Therefore, five domain swap protein constructs were generated with each of the three LIN12 repeats and the two dimerization segments replaced by the corresponding domains of Notch1. The domain swap constructs were generated using PCR-SOE (Ho, et al., Gene 77:51 (1989); Horton, et al., BioTechniques 8:528 (1990)) as illustrated in FIG. 12. PCR and PCR-SOE reactions were performed using PCR with 1M Betaine and 5% DMSO added to the reaction. PCR thermocycling was almost same for PCR and PCR-SOE except that the annealing step of each PCR cycle was extended one minute in PCR-SOE. The final PCR-SOE product was subcloned and verified by sequencing. The plasmid clone with the correct insert sequence was cleaved with Nhe I and Xho I to excise the insert, which was gel-purified and subcloned. The five Notch3/Notch1 domain swap constructs are illustrated in FIG. 12. To facilitate the epitope mapping, the human IgG kappa chain signaling peptide was used as leader peptide in the domain swap constructs. The amino acid sequences are shown in FIGS. 16 and 17.
Notch1-LD cDNA was PCR-amplified using PCR and methods described in the above section. The first strand cDNA template was synthesized from PA-1 cell total RNA (ATCC No. CRL-1572). The human IgG kappa chain leader peptide coding sequence was PCR-amplified, used as leader peptide to link to the 5′ of Notch1-LD by PCR-SOE and subcloned in His-γ1Fc/pSec.
Based on ELISA analysis results, target domains L1, D1 and D2 were further divided into subdomains. ELISA binding analysis using the subdomain expression constructs showed that only L1 and D2 were required for the Notch3 MAb binding. The D1 domain was not required. Therefore, L1 and D2 domains were divided into clusters of amino acid mutations for further analysis of the specific binding site. Constructs containing L1 and D2 subdomain swap or clusters of amino acid mutations as shown in FIG. 16 and FIG. 17 were generated.
C. Expression of Notch3/Notch1 Domain Swap Fusion Protein
Notch3/Notch1-LD domain swap plasmids were transiently transfected in CHO cells using LIPOFECTAMINETM 2000 transfection system. CHO cells were seeded in DMEM growth medium with 10% FCS at 0.8-1×106 cells per well in 6-well plate, maintained in CO2 incubator overnight before transfection. The cells were recovered after transfection in the growth medium for about 3 hours, then switched to DMEM with 2% FCS, and cultured for three days. The conditioned media were harvested and centrifuged at 3500 rpm for 10 minutes. The supernatant containing Notch3-LD domain swap protein secreted from CHO was collected and prepared for Western blot and ELISA binding analyses. ELISA showed that all the domain-swap fusion proteins were expressed and secreted in conditioned medium (Table 4), which was further confirmed by Western blot analysis (data not shown).
The ELISA readings used anti-human Fc antibody as detection antibody showing all the proteins were expressed in conditioned medium. Human IgG/Fc was used as a control. The starting point of human IgG/Fc coated in each well is 100 ng.







TABLE 4







ELISA Readings















Dilution
N1-LD
N3-LD
L1-swap
L2-swap
L3-swap
D1-swap
D2-swap
hIgG-Fc










Statistics: mean















1
3.2000
3.3445
3.4380
3.0970
3.2910
3.2870
3.4110
3.5510


0.250000
3.1305
2.7625
2.9890
2.7390
2.9050
3.0225
2.9570
3.4995


0.062500
2.3785
1.3870
2.8145
1.2835
2.6855
2.2575
2.3240
3.5805


0.015625
1.0085
0.3960
1.5245
0.3865
1.7350
0.9110
0.8800
3.2355


0.003906
0.3300
0.1075
0.4755
0.1220
0.5970
0.3450
0.2130
1.8585


0.000977
0.2095
0.0400
0.1640
0.1105
0.1780
0.1635
0.0615
0.5865


0.000244
0.1340
0.0225
0.0500
0.0595
0.0575
0.1045
0.0275
0.1445


6.104E−05
0.1000
0.0135
0.0405
0.0505
0.0230
0.0575
0.0305
0.0315


1.526E−05
0.0975
0.0165
0.0205
0.0430
0.0180
0.0400
0.0155
0.0220


3.815E−06
0.0580
0.0140
0.0135
0.0300
0.0150
0.0425
0.0235
0.0230


9.537E−07
0.0540
0.0125
0.0155
0.0245
0.0215
0.0480
0.0145
0.0165


2.384E−07
0.0415
0.0125
0.0145
0.0305
0.0155
0.0370
0.0150
0.0190







Statistics: S.D.















1
0.0778
0.0290
0.0679
0.0255
0.0933
0.1018
0.0283
0.0071


0.250000
0.0191
0.0304
0.0354
0.0396
0.0693
0.1619
0.1202
0.0148


0.062500
0.0898
0.0919
0.0007
0.1096
0.0318
0.0021
0.0071
0.0290


0.015625
0.0474
0.0354
0.0106
0.0417
0.1075
0.0071
0.0325
0.1450


0.003906
0.0523
0.0177
0.0460
0.0113
0.0453
0.0339
0.0057
0.0573


0.000977
0.0092
0.0057
0.0042
0.0191
0.0156
0.0205
0.0007
0.0955


0.000244
0.0226
0.0092
0.0014
0.0106
0.0064
0.0035
0.0049
0.0276


6.104E−05
0.0113
0.0007
0.0064
0.0035
0.0057
0.0134
0.0064
0.0064


1.526E−05
0.0021
0.0035
0.0049
0.0042
0.0000
0.0028
0.0007
0.0028


3.815E−06
0.0113
0.0028
0.0021
0.0000
0.0042
0.0064
0.0007
0.0057


9.537E−07
0.0014
0.0007
0.0007
0.0007
0.0064
0.0057
0.0021
0.0078


2.384E−07
0.0120
0.0035
0.0049
0.0021
0.0007
0.0113
0.0014
0.0127









Abbreviations for proteins used in the ELISA binding assays of Table 4 include: N1-LD, Notch1-LD/Fc. N3-LD, Notch3-LD/Fc. L1-swap: 1st LIN12 domain swap. L2-swap: 2nd LIN12 domain swap. L3-swap: 3rd LIN12 domain swap. D1-swap: 1st dimerization domain swap. D2-swap: 2nd dimerization domain swap. hIgG-Fc, human IgG Fc.
D. Epitope Binding Analysis Using ELISA
The 96-well flat bottom IMMULON II microtest plates (Dynatech Laboratories, Chantilly, Va.) were coated with anti-human Fc antibody (Jackson ImmunoResearch) by adding 100 μl of the antibody (0.1 μg/ml) in phosphate buffered saline (PBS) containing 1× Phenol Red and 3-4 drops pHix/liter (Pierce, Rockford, Ill.), and incubated overnight at room temperature. After the coating solution was removed by flicking of the plate, 200 μl of blocking buffer containing 2% BSA in PBST and 0.1% merthiolate was added to each well for one hour to block non-specific binding. The wells were then washed with PBST. Fifty microliters of the above conditioned medium from each transfection of Notch3/Notch1 domain swap construct were collected, mixed with 50 μl of blocking buffer, and added to the individual wells of the microtiter plates. After one hour of incubation, the Notch3/Notch1-LD domain swap protein was captured by the coated anti-Fc antibody, and the wells were washed with PBST. Anti-Notch3 MAbs and isotype-matched control MAbs were serially diluted in blocking buffer as above, and 50 μl of the diluted MAbs were added in each well to assess binding to the bound Notch3/Notch1 domain swap protein. Horseradish peroxidase (HRP)-conjugated, Fc-specific goat anti-mouse IgG was used for detection. HRP substrate solution containing 0.1% 3,3,5,5-tetramethyl benzidine and 0.0003% hydrogen peroxide was added to the wells for color development for 30 minutes. The reaction was terminated by addition of 50 ml of 2 M H2SO4/well. The OD at 450 nm was read with an ELISA reader. Subdomain swap constructs and clusters of mutations were similarly examined by ELISA analysis above.
ELISA binding experiments using MAbs 256A-4 and 256A-8 against the domain-swap proteins showed that the swap of the 1st LIN12 domain (L1) and 2nd dimerization domain (D2) completely abolished all the three MAbs binding, while the swap of 1st dimerization domain (D1) abolished binding of MAbs 256A-4 and 256A-8 (FIGS. 13 B&C). Swap of the 3rd LIN12 domain (L3) significantly weakened the binding. Nevertheless, both MAbs were still able to bind to the fusion protein. The swap of the 2nd LIN12 domain had no interference with the binding of the MAbs (FIGS. 13B and C). A positive control antibody, which was previously mapped to bind to the 1st LIN12 domain, bound to all domain swap fusion protein except L1 (FIG. 13D). In contrast, isotype control negative antibody, G3, does not bind to any of the domain swap fusion proteins in the ELISA assay (data not shown). It was concluded from the above experiments that the 1st LIN12 domain and 2nd dimerization domain were required for MAbs 256A-4 and 256A-8 binding.
To further map the epitopes in the 1st LIN12 domain (L1) to which anti-Notch3 MAbs bind, the L1 domain was further divided into three subdomains, L1-sub1, L1-sub2 and L1-sub3, and swapped with the corresponding sequences in Notch1 (FIG. 16). An ELISA binding assay showed that L1-sub1 swap has no inhibitory effects on binding activity, and L1-sub2 and L1-sub3 swap abolished binding (FIG. 16). In L1-sub2 and L1-sub3 regions, there are five clusters of amino acid residues that differ between Notch3 and Notch1. Therefore, swap fusion protein constructs were generated within these five clusters of amino acids (FIG. 16). ELISA analysis demonstrated that L1-cluster4 swap had no inhibition on all three MAbs binding. The remaining four clusters of swap partially or completely abolished the anti-Notch MAbs binding. Thus, those four clusters of amino acid residues represented four different epitopes to which the MAbs bind. L1-cluster3 (amino acids: DRE) and L1-cluster5 (amino acids: SVG) are required. L1-cluster1 (amino acids: AKR) and cluster2 (amino acids: DQR) also played a role in anti-Notch3 MAb binding, whose mutations significantly weakened the MAb binding.
To map the epitopes in the 2nd dimerization (D2) domain of Notch3 to which anti-Notch3 MAbs bind, the D2 domain was further divided into five subdomains, D2-sub1, D2-sub2, D2-sub3, D2-sub4 and D2-sub5. The sequences in those subdomains were swapped with the corresponding sequences in Notch1 (FIG. 17). An ELISA binding assay showed that MAbs 256A-4 and 256A-8 have strong binding to D1-sub2 and D2-sub3 swap, but not to D2-sub1 and D2-sub4 swap. Both MAbs showed weak binding to D2-sub5 (FIG. 17). Therefore, the data suggested that D2-sub1 and D2-sub4 are required for the anti-Notch3 MAb binding and D2-sub5 may help the binding activity.
Both MAbs 256A-4 and 256A-8 are antagonistic antibodies binding to the conformational epitope comprising L1 and D2, while another antibody 256A-13 that binds only to L1 is agonistic (See co-pending U.S. application Ser. No. 11/874,682, filed Oct. 18, 2007). Furthermore, agonistic 256A-13 competes with antagonistic 256A-4 for an epitope within L1, and the epitope mapping studies suggest that they bind to an overlapping epitope on L1. The major difference is that the antagonistic antibodies also bind to D2, while the agonistic antibody does not. To test the hypothesis that simultaneous binding to L1 and D2 is responsible for the antagonistic activity, an antibody, 256A-2 binding to a similar epitope in D2 as 256A-4 was analyzed. MAb 256A-2 is neither antagonistic nor agonistic (data not shown). Studies showed that 256A-2 does not compete with 256A-13 and can bind to Notch3 simultaneously. Furthermore, 256A-2 and 256A-13 individually can partially compete with 256A-4, however, in combination these two antibodies completely block binding of 256A-4 to Notch3 (data not shown). Studies also showed that separate binding of two antibodies to the epitopes in L1 and D2 does not lead to the inhibition of ligand-dependent Notch3 activation, suggesting that the antagonistic antibodies form a bridge, possibly locking and stabilizing the L1 and D2 interaction, and preventing the ligand induced conformational changes. (See FIG. 18)
Example 9
Sequencing of Anti-Notch3 MAbs
Because antibody binding properties are dependent on the variable regions of both heavy chain and light chain, the variable sequences of 256A-4 and 256A-8 were subtyped and sequenced. The antibody IgG subtype was determined using an ISOSTRIPTM mouse monoclonal antibody isotyping kit (Roche Diagnostics, Indianapolis, Ind.). The results showed that both MAbs, 256A-4 and 256A-8 have an IgG1 heavy chain and a kappa light chain.
The variable region sequences of heavy chain and light chain were decoded through RT-PCR and cDNA cloning. Total RNAs from hybridoma clones 256A-4 and 256A-8 were isolated using an RNeasy Mini kit following the manufacturer's protocol (QIAGEN, Valencia, Calif.). The first strand cDNA was synthesized using the RNA template and SUPERSCRIPT® III reverse transcriptase kit. The variable region of light chain and heavy chain cDNAs were PCR-amplified from the first strand cDNA using degenerative forward primers covering the 5′-end of mouse kappa chain coding region and a reverse primer matching the constant region at the juncture to the 3′-end of the variable region, or using degenerative forward primers covering the 5′-end of mouse heavy chain coding region and a constant region reverse primer in mouse heavy chain. The PCR product was cloned into a commercially available vector and sequenced by Lone Star Lab (Houston, Tex.). The nucleotide sequences were analyzed utilizing the DNASTAR® computer software program (DNASTAR, Inc., Madison, Wis.). Each anti-Notch3 MAb sequence was determined by sequences from multiple PCR clones derived from the same hybridoma clone.
MAb 256A-4 contains 123 and 116 amino acid residues, respectively, in its variable region of heavy chain and light chain (FIGS. 4A and 4B). MAb 256A-8 consists of 122 and 123 amino acid residues in heavy chain and light chain variable regions, respectively (FIGS. 5A and 5B).
Example 10
Impact of Notch3 Antagonistic Antibodies on Metalloprotease Cleavage of Notch3
Notch receptor activation involves ligand induced metalloprotease cleavage at juxtamembrane site (S2) generating an extracellular subunit. This cleavage is an essential prerequisite to S3 cleavage to release the activated Notch intracellular region. Both 256A-4 and 256A-8 were found to require the presence of at least a portion of the Notch3 L1 and D2 domains for their bindings. These two domains are not located in close proximity in the linear sequence, but rather are on two separate polypeptides, suggesting these antibodies may stabilize an inactive, autoinhibited Notch configuration. To test whether the antagonizing antibodies can inhibit sequential Notch activation events, including two proteolytic cleavages, 293T cells stably expressing a recombinant Notch3 receptor (NC85 cells) are treated with either immobilized recombinant Jagged-1 or cocultured with 293T cells expressing Jagged-1. The soluble extracellular subunits generated by proteolytic cleavage in the culture medium are detected by an ELISA assay using an antibody bound to a solid surface that recognizes the Notch3 cleavage product. Notch3 antagonistic MAbs are expected to decrease the generation of soluble Notch3 extracellular subunits in the conditioned medium, whereas non-functional Notch3 binding antibodies would not.
To directly detect the S2 cleavage fragment, an 7.5% SDS PAGE electrophoresis and Western blot with Notch3 C-terminal antibody are performed. The S2 fragment is 57 amino acids residues smaller and migrates slightly faster than the non-cleaved Notch3 small subunit (transmembrane subunit).
To examine whether Notch3 antagonistic MAbs inhibit ligand-induced metalloprotease cleavage of Notch3 at S2, 293T cells expressing recombinant Notch3 were treated with the γsecretase inhibitor compound E (1 μM) for 4 hours, which stabilizes the product of cleavage at site S2, allowing it to accumulate. In the presence of MAbs 256A-4 and 256A-8, Jagged-1-induced metalloprotease cleavage of Notch3 at S2 was inhibited (data not shown).
Example 11
Efficacy Study Using Human Cancer Models in Xenograft Mice
A. Human Cancer Cells and Tumorigenic Cells
Human cancer cell lines with Notch3 expression such as HCC2429, HCC95 may be obtained from Academic Institutes, or from the ATCC. The 293T/PCDNATM 3.1, and 293T/Notch3 (NC85) cells are generated by transfecting 293T with related genes and selecting with hygromycin as describe in previous sections. All cells are cultured in DMEM or RPMI 1640 medium with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, vitamin solution, and penicillin-streptomycin (Flow Laboratories, Rockville, Md.). Cell lines are incubated in a mixture of 5% CO2 and 95% air at 37° C. in an incubator. Cultures are maintained for no longer than 3 weeks after recovery from frozen stocks. Logarithmically growing single-cell suspensions cells with ≧90% viability are used for tumor cells injection after washing with PBS.
B. Animals
Mice are obtained from, for example, the Animal Production Area of the National Cancer Institute at Frederick Cancer Research and Development Center, Frederick, Md. The animals are purpose-bred and are experimentally naïve at the outset of the study. Mice selected for use in the studies are chosen to be as uniform in age and weight as possible. They are 6-8 weeks of age and their body weights at initiation of weight range from approximately 18 to 25 grams. Records of the dates of birth for the animals used in this study are retained in the study raw data, and the weight range at the time of group assignment is specified in the report. Each animal is identified by a numbered ear tag. The animals are group housed by treatment group (4 mice/cage) in polystyrene disposable shoe-box cages containing cellulose bedding, meeting or exceeding NIH guidelines. During the course of the study, the environmental conditions in the animal room is monitored and maintained within a temperature range of 18-26° C., and the relative humidity is recorded daily. A 12-hour light/dark illumination cycle is maintained throughout the study. Animals have irradiated food. No contaminants are known to be present in the food at levels that would interfere with the results of this study. Autoclaved water is available to each animal via water bottles. No contaminants are known to be present in the water at levels that would interfere with the results of this study. Prior to assignment to the study, all study animals are acclimatized to their designated housing for at least 7 days prior to the first day of dosing.
C. Tumor Models and Efficacy Studies
Mice are anesthetized using sodium pentobarbital (50 mg/kg body weight) and placed in the right lateral decubitus position. Cancer cells, such as non-small cell lung cancer (NSCLC) cell lines, HCC2429 (Haruki, et al. Cancer Res. 65:3555 (2005)), HCC95 (From Dr. John Mina), and H2122 (ATCC No. CRL5985), in 50 μl Hank's containing 10% MATRIGELTM matrix are injected into the left lobe of the lungs. After the tumor-cell injection, the mice are turned to the left lateral decubitus position and observed for 45-60 min until they recover fully. Records of tumor cell injections are maintained in the raw study data.
All animals are observed within their cages at least once daily during study and clinical findings recorded in the study raw data. Animals that show pronounced detrimental effects may be removed from the study should it be deemed necessary. Body weight is measured once each week during the treatment. Cancer tissues from each mouse, where available, are harvested and stored for potential future biological characterization.
Example 12
Assay for Notch3 Related Diseases
To identify other Notch3 related diseases, one can sequence the Notch3 gene from patient samples, perform FISH (fluorescence in situ hybridization) and CGH (comparative genomic hybridization) analysis to look for translocation and gene amplification using patient cells, or perform immunohistochemistry to check for the over-expression of Notch3 receptor using patient tissue or tumor sections. In addition, one can isolate and culture cells from a patient suspected of having a Notch3 associated disease and study the impact of an antagonistic antibody of the present invention on cell migration, invasion, survival and proliferation. Protocols for cell migration and invasion assay are described in Example 7 and the protocol for an apoptosis assay is described in Example 6. For the cell proliferation assay, cells cultured from patient samples are be seeded in 96-well plate coated with and without Notch ligands. Antagonistic antibodies are added at the beginning of the culture. Cell numbers are counted at specific time points using trypan blue staining. Notch3 FISH and CGH analysis may be performed using the published protocols of Park, et al. (Cancer Res, 66: 12 (2006)).
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

## Claims

1. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of the VH chain region of a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.

2. The nucleic acid of claim 1, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).

3. The nucleic acid of claim 1, wherein the VH chain region comprises CDR-H1 of SEQ ID NO:32, CDR-H2 of SEQ ID NO:33, and CDR-H3 of SEQ ID NO:34.

4. The nucleic acid of claim 3, wherein the VH chain region comprises SEQ ID NO:2.

5. The nucleic acid of claim 1, wherein the VH chain region comprises CDR-H1 of SEQ ID NO:38, CDR-H2 of SEQ ID NO:39, and CDR-H3 of SEQ ID NO:40.

6. The nucleic acid of claim 5, wherein the VH chain region comprises SEQ ID NO:4.

7. An isolated cell comprising the nucleic acid of claim 1.

8. A method for producing a polypeptide comprising the amino acid sequence of the VH chain region of a monoclonal antibody that specifically binds to Notch3 comprising culturing the cell of claim 7 under conditions appropriate for production of a polypeptide and isolating the polypeptide produced.

9. An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of the VL chain region of a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.

10. The nucleic acid of claim 9, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).

11. The nucleic acid of claim 9, wherein the VL chain region comprises CDR-L1 of SEQ ID NO:35, CDR-L2 of SEQ ID NO:36, and CDR-L3 of SEQ ID NO:37.

12. The nucleic acid of claim 11, wherein the VL chain region comprises SEQ ID NO:3.

13. The nucleic acid of claim 9, wherein the VL chain region comprises CDR-L1 of SEQ ID NO:41, CDR-L2 of SEQ ID NO:42, and CDR-L3 of SEQ ID NO:43.

14. The nucleic acid of claim 13, wherein the VL chain region comprises SEQ ID NO:5.

15. An isolated cell comprising the nucleic acid of claim 9.

16. A method for producing a polypeptide comprising the amino acid sequence of the VL chain region of a monoclonal antibody that specifically binds to Notch3 comprising culturing the cell of claim 15 under conditions appropriate for production of a polypeptide and isolating the polypeptide produced.

17. An isolated nucleic acid encoding a monoclonal antibody that specifically binds to Notch3, wherein the antibody specifically binds to a conformational epitope of a Notch3 fragment consisting of amino acids 1378-1640 of SEQ ID NO:1, and wherein the antibody inhibits Notch3 signaling.

18. The nucleic acid of claim 17, wherein the antibody binds to amino acid residues in the LIN12 domain (SEQ ID NO:9) and the dimerization domain (SEQ ID NO:18).

19. The nucleic acid of claim 17, wherein the antibody comprises a VH chain region comprising CDR-H1 of SEQ ID NO:32, CDR-H2 of SEQ ID NO:33, and CDR-H3 of SEQ ID NO:34, and a VL chain region comprising CDR-L1 of SEQ ID NO:35, CDR-L2 of SEQ ID NO:36, and CDR-L3 of SEQ ID NO:37.

20. The nucleic acid of claim 19, wherein the VH chain region comprises SEQ ID NO:2 and the VL chain region comprises SEQ ID NO:3.

21. The nucleic acid of claim 17, wherein the antibody is a humanized form of a monoclonal antibody comprising the VH chain region of SEQ ID NO:2 and the VL chain region of SEQ ID NO:3.

22. The nucleic acid of claim 17, wherein the antibody comprises a VH chain region comprising CDR-H1 of SEQ ID NO:38; CDR-H2 of SEQ ID NO:39, and CDR-H3 of SEQ ID NO:40, and a VL chain region comprising CDR-L1 of SEQ ID NO:41; CDR-L2 of SEQ ID NO:42, and CDR-L3 of SEQ ID NO:43.

23. The nucleic acid of claim 22, wherein the VH chain region comprises SEQ ID NO:4 and the VL chain region comprises SEQ ID NO:5.

24. The nucleic acid of claim 17, wherein the antibody is a humanized form of a monoclonal antibody comprising the VH chain region of SEQ ID NO:4 and the VL chain region of SEQ ID NO:5.