# Patent

## Title
Antibody-like proteins for therapeutic and diagnostic use

## Abstract

Disclosed herein are recombinant protein scaffolds for use in producing antigen-binding proteins. Related antigen-binding proteins are also provided herein. In addition, nucleic acids encoding such recombinant protein scaffolds and antigen-binding proteins are also described. Vectors and cells useful for expression of the described proteins are also provided, as are methods of use.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the National Stage of International Application No. PCT/US2012/028286, filed Mar. 8, 2012, which claims the benefit of U.S. Provisional Application No. 61/450,435 filed Mar. 8, 2011, the disclosures of which are incorporated herein by reference in their entireties for any and all purposes.


GOVERNMENT RIGHTS
The subject matter disclosed herein was made with government support under award/contract/grant No. CA055306 awarded by the National Institutes of Health. The Government hasa certain rights in the herein disclosed subject matter.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 24, 2015, is named 103241.005883-X5822_SL.txt and is 44,371 bytes in size.
TECHNICAL FIELD
The present invention relates to peptide-Fc fusion proteins that can be used in detection assays, as therapeutics or as substitutes for monoclonal antibodies in other applications.
BACKGROUND
Since hybridoma technology enabled long-lived hybridoma production of monoclonal antibodies in the mid-1970's, scientist and clinicians have been trying to harvest their therapeutic potential to treat diseases. Over the years, science has pushed back the frontiers of antibody technology, allowing for the development of chimeric antibodies, humanized antibodies, and immunologically functional antibody fragments, such as Fabs and diabodies. Today, there are numerous antibody therapeutics used to treat diseases such as cancer, infectious diseases, and autoimmune disorders, just to name a few. In addition to existing therapeutics, more are on the horizon and the scientific community is working feverishly to develop new and/or more effective antibody-based therapeutics.
While antibody therapeutics have proven successful in recent years, with at least 25 such therapeutics having gained FDA approval, they are not without drawbacks. Some drawbacks to antibodies include their large size (approximately 150 kD) and that they often require proper post-translational processing. The large size of antibodies can reduce their ability to target certain diseases, such as cancer or neurological disorders, which may require crossing the blood-brain barrier. The fact that many antibodies require proper post-translational processing by a eukaryotic cell often requires that antibody therapeutics be produced in, and subsequently purified from, mammalian cell culture, which can hinder total antibody production and increase production costs, relative to proteins produced in bacteria.
To overcome some of the drawbacks of antibodies, non-antibody synthetic proteins have been developed. Some examples of non-antibody synthetic proteins include antibody fragments, such as Fabs, scFvs, diabodies, Affibodies®, and Nanobodies®, to name a few. Proteins such as these, while smaller than antibodies and useful for some applications, often do not have the ability to interact with antibody receptors, such as the Fc receptor, expressed by immune effector cells, which can enhance the activity of the immune system.
To address some of the shortcomings of antibodies and non-antibody synthetic proteins, disclosed herein are antigen-binding protein constructs that incorporate all or part of an antibody Fc domain.
SUMMARY
Described herein are protein scaffolds for use in producing antigen-binding proteins. A variety of protein scaffolds are described, as are a variety of functional attributes or characteristics that can be associated with the described protein scaffolds. Several of the disclosed protein scaffolds are made of at least one protein scaffold or framework segment that can be linked to at least one antigen-specific polypeptide sequence, known as an antigen-specific peptide, to form an antigen-binding protein. The scaffold proteins described herein can also be modified to bind, or enhancing binding, to the fragment crystallizable (Fc) receptors. In addition, some of the scaffold protein segments may inherently possess these, or other, desired functional characteristics, which also allows them to be attached to other antigen-specific proteins to confer this activity. Some of the antigen-binding proteins resulting from the combination of the described scaffold protein constructs and an antigen-specific peptide that are described herein have the ability to bind a particular antigen and an Fc receptor in addition to having an attached detectable label. Also disclosed are polynucleotides encoding the described protein scaffolds or exemplary antigen-binding proteins, vectors encoding the described scaffolds or exemplary antigen-binding proteins, cells transformed with the disclosed vectors, and methods of treating, diagnosing, or preventing disease using the described antigen-binding proteins, and methods of detecting antigens of interest using the described antigen-binding proteins.
One embodiment of the protein scaffolds described herein can be all or a portion of a heavy chain constant region 2 (CH2) of an antibody, for example, SEQ ID NO:22. The described CH2 region can be linked or combined with at least one antigen-specific peptide to form an antigen-binding protein. In one embodiment, the antigen-binding protein includes the CH2 scaffold linked to an antigen-specific peptide, such as the S22 peptide (SEQ ID NO:15) derived from human epidermal growth factor receptor 2 (HER2) receptor, to create an antigen-binding protein that is able to bind HER2, epidermal growth factor receptor (EGFR), or human epidermal growth factor receptor 3 (HER3). In another embodiment this antigen-binding protein, or an analogous antigen-binding protein specific for a different antigen, can include a detectable label, for example, an epitope tag, a fluorophore, a radio isotope, or an enzyme. It should be noted that many embodiments of the antigen-binding proteins described herein are exemplified using the antigen-specific peptide S22; however, any of the antigen-specific peptides known to those of skill in the art, can be used to produce antigen-binding proteins using the protein scaffolds described herein. Antigen-specific peptides that may be used with the described protein scaffolds to produce antigen-binding proteins include those associated with SEQ ID NOs:24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.
The described CH2 region can also be conjugated to a corresponding CH3 region (such as that exemplified by SEQ ID NO:23) and an antigen-specific peptide, to form an antigen-binding protein that includes, or approximates, the Fc domain of an antibody. One embodiment of such an antigen-binding protein can include the S22 peptide fused to a CH2 region, which is also attached to a CH3 region. One such embodiment is provided by the amino acid sequence of, or substantially similar to, SEQ ID NO:1. The CH2 and CH3 regions described herein can be obtained from any adequate antibody having such regions, for example IgG, IgD, IgM, IgE, IgA, or IgY, to name several. The sources of such antibodies can vary also, such that these regions could be obtained from a human antibody, murine antibody, primate antibody, rodent antibody, avian antibody or other such vertebrate source. In some embodiments, a particular subtype of antibody may be used, such as IgG1. In other embodiments, the CH2 and CH3 regions maybe mixed. For example, a CH2 from a human IgG1 antibody could be joined with a CH3 region of a murine IgG2 antibody. Other such combinations analogous to this example would be apparent to those skilled in the art. It should be noted that many embodiments of the antigen-binding proteins described herein are often exemplified using the antigen-specific peptide S22; however, any of the antigen-specific peptides known to those of skill in the art, can be used to produce antigen-binding proteins using the protein scaffolds described herein.
The described protein scaffolds and antigen-binding proteins can also include altered amino acid sequences that provide an enhanced protein function or reduce one or more undesirable property of the construct. These alterations may be achieved by a number of techniques known in the art, such as genetic engineering to alter the resulting amino acid sequence of the antigen-binding protein, chemical modification of the antigen-binding protein, or the addition functional peptides or motifs. In one embodiment, the described CH2 region, or Fc domain, may be altered to include one of more mutated amino acid residues. For example, residue 299 of the CH2 domain (or the amino acid corresponding to this residue in a related antigen-binding protein) may be modified to be a different amino acid. In one embodiment, residue 299 of the CH2 domain is changed from threonine to alanine. It will be understood by those skilled in the art that amino acid 299 of the CH2 region will not necessarily appear at position 299 in instances where a CH2 region is modified or incorporated to a larger scaffold or antigen-binding protein. Accordingly, in some embodiments, a residue of a protein-binding protein construct incorporating a CH2 region may be modified to encode a residue other than threonine at the amino acid corresponding to residue 299 of the CH2 region. For example, in one embodiment, a mutation of the residue corresponding to amino acid 299 of the CH2 region may occur at position 322 for an antigen-binding protein construct where the S22 peptide is linked to a CH2 region, since the addition of the S22 peptide adds N-terminal residues to the CH2 region. While this example is provided in the context of the CH2 peptide, other antigen specific peptides could alter the positioning of the CH2 residues in an analogous manner. Other scaffolds provided herein include LFcG7 (SEQ ID NO:16), LFcT322AG7 (SEQ ID NO:17), LFcT322AS7 (SEQ ID NO:18), and MFcG7 (SEQ ID NO:19) and MFcT322AG7 (SEQ ID NO:20).
Also disclosed herein are the amino acid sequences for a number of the scaffolds and antigen-binding proteins described, as such, corresponding nucleotide sequences encoding these scaffolds and antigen-binding proteins will be apparent to those skilled in the art. Vectors used to express these polynucleotide sequences encoding the disclosed amino acid sequences are also provided. For the sake of brevity, only a limited number of vectors having polynucleotide sequences capable of encoding the described constructs are provided; however, alternative vector and polynucleotide combinations for expressing the disclosed antigen-binding proteins will be apparent to those with sufficient skill in the art to understand the degeneracy of the genetic code. Additionally, it is fully contemplated that the disclosed vectors can be used to transform prokaryotic and/or eukaryotic cells to facilitate expression of the described antigen-binding proteins. In some embodiments the described vectors are used to facilitate protein expression in bacteria, such as E. coli. While any E. coli strain can be used to express the proteins described herein, some preferred strains include: BL21 (DE3), BL21-CodonPlus® (DE3)-RP, BL21-CodonPlus® (DE3)-RIL, BL21-(DE3)-pLysS (Stratagene). Eukaryotic cells can also be used with the described vectors to facilitate protein expression. While those of skill in the art will recognize that a wide variety of eukaryotic cells will be suitable for this purpose, some preferred embodiments include mammalian cells and insect cells. For example, in one embodiment Chinese hamster ovary (CHO) cells can be used with the described vectors to facilitate expression of the protein constructs provided herein. In alternative embodiments, insect cells, such as Sf9 cells or S2 cells, can be used to with the described vectors to facilitate expression of the protein constructs provided herein. Furthermore, those of skill in the art will understand that alternative vectors, not expressly disclosed herein, can be used for the same purpose of expressing, or replicating nucleic acids encoding, the described antigen-binding proteins.
Also described herein are compositions containing an antigen-binding protein and a pharmaceutically acceptable carrier. Such compositions can be used to administer the described antigen-binding proteins to a subject or store or to maintain the described antigen-binding proteins. Any of the described antigen-binding proteins can be used to produce such compositions, which may include more than one of the disclosed antigen-binding proteins. In addition, such compositions can include other agents, such as therapeutic agents, preservatives, antimicrobial agents, and the like.
Methods of using the described scaffolds, or antigen-binding proteins, are also provided. For example, antigen-binding proteins derived from the scaffolds disclosed herein may be used to treat or prevent disease in a subject. The described methods of treating or preventing disease can be used to administer compositions, having antigen-binding proteins derived from the described scaffolds, to a subject in need of such treatment. Also disclosed are methods for detecting an antigen of interest using antigen-binding proteins derived from the scaffolds disclosed herein. Such methods are applicable to antigen detection in a subject, in a sample obtained from a subject, or in vitro.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a schematic representation of the S22-Fc antigen-binding protein construct. Amino acid sequences for the S22 peptide and glycine-serine linker are shown (residues 1-19 of SEQ ID NO:1).
FIG. 2 shows gel-resolved expression of the S22-Fc fusion proteins in BL21(DE3) codon plus (Stratagene, Inc). Expression of recombinant proteins was induced by 0.5 mM IPTG at either 37° C. or 16° C. as indicated. UI: uninduced cells; P: inclusion body of induced cells containing insoluble proteins; S: Soluble proteins from induced cells.
FIG. 3 shows the results of FACS experiments to stain cells (T6-17, NE91, and MCF7) with different expression levels of HER2 and EGFR with purified S22-Fc.
FIG. 4 shows FACS analysis of T6-17, NE91, MCF7, and MM3MG cells stained with antigen-binding proteins S22-Fc, S22-FcT322A, S22-Fc322#2, S22-Fc322#4.
FIG. 5 depicts the results binding of several LS22-Fc constructs to the T6-17 cell line as assessed by FACS analysis. The geometric mean of each construct is also listed. The solid grey peak represents the control cells that were stained only with anti-his tag and secondary antibodies.
FIG. 6 shows inhibition of tumor growth in athymic mice carrying HER2-positive T6-17 xenografts treated with either LS22FcT322AG7, the 4D5 antibody, or buffer.
FIG. 7 shows inhibition of tumor growth in athymic mice carrying EGFR-positive A431 xenografts treated with either LS22Fc, LS22FcT322AG7, or buffer.
FIGS. 8a-8d provide illustrations of the vector design strategy for the antigen-binding protein constructs S22-Fc (8a), S22-FcT322A#4 (8b), LS22-Fc (8c), LS22-FcT322A (8d).
FIG. 9 shows binding of the HER2-specific antibody 7.16.4 to LS22FcT322AG7 (A) and that interaction of the murine HER2-specific antibody 4D5 with HER2 is inhibited in the presence of LS22FcT322AG7 (B). In subpart (B), excluding the left-most control peak, the right-most peak represents binding of m4D5 alone, the center peak represents binding of m4D5 in the presence of LS22FcT322AG7 (10 μg), and the left peak represents binding of m4D5 in the presence of LS22FcT322AG7 (50 μg).
FIG. 10 depicts a Western blot that show reduced EGF-induced phosphorylation of HER3 in the presence of LS22FcT322AG7 (A), a Western blot that show reduced HRG-induced phosphorylation of HER3 in the presence of LS22FcT322AG7 or an anti-HER3 antibody (positive control) (B), reduced in vivo HCl tumor growth in the presence of LS22FcT322AG7, and (C) HCl xenographs implanted subcutaneously into athymic mice.
FIG. 11 shows and reduced in vivo TB129 tumor growth in the presence of LS22FcT322AG7.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
As used herein, the term “cytotoxic” or “cytostatic” agent refers to an agent that inhibits the biological processes of a cell, or reduces the viability or proliferative potential of a cell. Cytotoxic or cytostatic agents can function in a variety of ways, for example, but not by way of limitation, by inducing DNA damage, inducing cell cycle arrest, inhibiting DNA synthesis, inhibiting transcription, inhibiting translation or protein synthesis, inhibiting cell division, or inducing apoptosis. As used herein, the term “chemotherapeutic agent” refers to cytotoxic, cytostatic, and antineoplastic agents that preferentially kill, inhibit the growth of, or inhibit the metastasis of neoplastic cells or disrupt the cell cycle of rapidly proliferating cells. Chemotherapeutic agents include, but are not limited to, synthetic compounds, natural and recombinant bacterial toxins, natural and recombinant fungal toxins, natural and recombinant plant toxins, fissionable nuclides, and radionuclides. Specific examples of chemotherapeutic agents include, but are not limited to, pokeweed antiviral protein, abrin, ricin and each of their A chains, momordin, saporin, bryodin 1, bouganin, gelonin, Diphtheria toxin, Pseudomonas exotoxin, Shiga toxin, calicheamicin, maytansinoid, lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111, boron-10, actinide, altretamine, actinomycin D, plicamycin, puromycin, gramicidin D, doxorubicin, colchicine, cytochalasin B, cyclophosphamide, emetine, maytansine, amsacrine, cisplastin, etoposide, etoposide orthoquinone, teniposide, daunorubicin, gemcitabine, doxorubicin, mitoxantraone, bisanthrene, Bleomycin, methotrexate, vindesine, adriamycin, vincristine, vinblastine, BCNU, taxol, tarceva, avastin, mitomycin, 5-fluorouracil, cyclophosphamide and certain cytokines such as TNF-alpha and TNF-beta.
“Polynucleotide,” synonymously referred to as “nucleic acid molecule” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
“Substantially the same” with respect to nucleic acid or amino acid sequences, means at least about 65% identity between two or more sequences. Preferably, the term refers to at least about 70% identity between two or more sequences, more preferably at least about 75% identity, more preferably at least about 80% identity, more preferably at least about 85% identity, more preferably at least about 90% identity, more preferably at least about 91% identity, more preferably at least about 92% identity, more preferably at least about 93% identity, more preferably at least about 94% identity, more preferably at least about 95% identity, more preferably at least about 96% identity, more preferably at least about 97% identity, more preferably at least about 98% identity, and more preferably at least about 99% or greater identity. Such identity can be determined using mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87:2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-7).
A “vector” is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
The term “operably linked” or “operably inserted” means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. By way of example, a promoter is operably linked with a coding sequence when the promoter is capable of controlling the transcription or expression of that coding sequence. Coding sequences can be operably linked to promoters or regulatory sequences in a sense or antisense orientation. The term “operably linked” is sometimes applied to the arrangement of other transcription control elements (e.g., enhancers) in an expression vector.
A cell has been “transformed” or “transfected” by exogenous or heterologous nucleic acids such as DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell, or “stable cell” is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
“Biomolecules” include proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof.
The terms “express” and “produce” are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications. The expression/production of an antibody or antigen-binding fragment can be within the cytoplasm of the cell, and/or into the extracellular milieu such as the growth medium of a cell culture.
The terms “treating” or “treatment” can refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment or amelioration of symptoms can be based on objective or subjective parameters.
“Effective amount” and “therapeutically effective amount” are used interchangeably herein, and refer to an amount of an antibody, antigen-binding fragment, or antibody composition, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, biological or therapeutic results disclosed, described, or exemplified herein. A therapeutically effective amount of the antibody or antigen-binding fragment thereof may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding fragment thereof to elicit a desired response in the individual. Such results may include, but are not limited to, the treatment of cancer, as determined by any means suitable in the art.
“Pharmaceutically acceptable” refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.
“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric and polymeric forms of each isotype, unless otherwise specified.
“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof, such as Fab, Fab′, F(ab′) 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Various techniques have been developed for the production of antibody fragments, including proteolytic digestion of antibodies and recombinant production in host cells; however, other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In some embodiments, the antibody fragment of choice is a single chain Fv fragment (scFv). “Single-chain Fv” or “scFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).
“Scaffold” refers to a recombinant polypeptide structure that can provide a framework to which another protein or polypeptide may be linked, or fused, to allow for increased stability of the protein or peptide or to place the protein or peptide in a more preferred conformation, or form, to mediate a desired biological activity.
“Linked” in the context of a scaffold or antigen-binding protein means “connected to” either directly or indirectly. Indirect linkage can be mediated by a polypeptide linker such as poly-glycine or a glycine-serine polypeptide, for example, GGGGGS (SEQ ID NO:21). Other such linkers are known in the art and should be considered to be encompassed by this term.
Described herein are protein scaffolds for use in producing antigen-binding proteins. A variety of protein scaffolds are described, as are a variety of functional attributes or characteristics that can be associated with the described protein scaffolds. Several of the disclosed protein scaffolds are made of at least one protein scaffold or framework segment that can be linked to at least one antigen-specific polypeptide sequence, known as an antigen-specific peptide, to form an antigen-binding protein. The scaffold proteins described herein can also be modified to bind, or enhancing binding, to the fragment crystallizable (Fc) receptors. In addition, some of the scaffold protein segments may inherently possess these, or other, desired functional characteristics, which also allows them to be attached to other antigen-specific proteins to confer this activity. Some of the antigen-binding proteins resulting from the combination of the described scaffold protein constructs and an antigen-specific peptide that are described herein have the ability to bind a particular antigen and an Fc receptor in addition to having an attached detectable label. Also disclosed are polynucleotides encoding the described protein scaffolds or exemplary antigen-binding proteins, vectors encoding the described scaffolds or exemplary antigen-binding proteins, cells transformed with the disclosed vectors, and methods of treating, diagnosing, or preventing disease using the described antigen-binding proteins, and methods of detecting antigens of interest using the described antigen-binding proteins.
The antigen-binding proteins exemplified herein have been made with the antigen-specific peptide S22, which allows for comparative studies between the scaffolds, and antibodies or antibody fragments that have similar antigen specificity. This practical aspect of the present disclosure should not be considered to limit the scaffolds provided herein for use with only S22, as it should be apparent that these scaffolds, as described in Table 1, can be used with a wide variety of antigen-specific peptides. While the particular amino acid sequences provided represent the specific embodiments exemplified herein, it should be understood that certain amino acid substitutions, deletions, or additions could be made to the described sequences that would not alter the function of the described scaffolds or antigen-binding proteins. Therefore, it is contemplated that scaffolds or antigen-binding proteins having at least 80% homology to those described herein are within the scope of the disclosed subject matter. Furthermore, it is contemplated that scaffolds or antigen-binding proteins having at least 85% homology to those described herein are within the scope of the disclosed subject matter. In addition, it is contemplated that scaffolds or antigen-binding proteins having at least 90% homology to those described herein are within the scope of the disclosed subject matter. Moreover, it is contemplated that scaffolds or antigen-binding proteins having at least 95% homology to those described herein are within the scope of the disclosed subject matter.
One embodiment of the protein scaffolds described herein is a heavy chain constant region 2 (CH2) of an antibody, such as SEQ ID NO:22. The described CH2 region can be linked or combined with at least one antigen-specific peptide to form an antigen-binding protein. In one embodiment, the antigen-binding protein includes the CH2 scaffold linked to an antigen-specific peptide, such as the S22 peptide (SEQ ID NO:15) derived from HER2, to create an antigen-binding protein that is able to bind HER2, EGFR, or HER3. In another embodiment this antigen-binding protein, or an analogous antigen-binding protein specific for a different antigen, can include a detectable label, for example, an epitope tag, a fluorophore, a radio isotope, or an enzyme. It should be noted that many embodiments of the antigen-binding proteins described herein are exemplified using the antigen-specific peptide S22; however, any of the antigen-specific peptides known to those of skill in the art, can be used to produce antigen-binding proteins using the protein scaffolds described herein.
The described CH2 region can also be conjugated to a corresponding CH3 region and an antigen-specific peptide, to form an antigen-binding protein. In some embodiments an antigen-binding protein with a CH2 and CH3 domain can include, or approximate, an Fc domain of an antibody. In some embodiments, the CH2 region may include one or more amino acid residues associated with the hinge region of an antibody. For example, a CH2 having hinge region amino acids may be used to allow the CH2 region to better approximate the CH2 region of an Fc domain. Such CH2 region may be used with or without a CH3 region to approximate an Fc domain. In one embodiment of the antigen-binding proteins described herein the S22 peptide is fused to all, or part of, the hinge region of an antibody preceding a CH2 region, which in turn is attached to a CH3 region. One embodiment of the antigen-binding proteins described herein can include the S22 peptide fused to a CH2 region, which is also attached to a CH3 region. One such embodiment is provided by the amino acid sequence of, or substantially similar to, SEQ ID NO:1. The CH2 and CH3 regions described herein can be obtained from any adequate antibody having such regions, for example IgG, IgD, IgM, IgE, IgA, IgY and any subtype thereof, to name several. The sources of such antibodies can vary also, such that these regions could be obtained from a human antibody, murine antibody, primate antibody, rodent antibody, avian antibody or other such vertebrate source. In some embodiments, a particular subtype of antibody may be used, such as IgG1. In other embodiments, the CH2 and CH3 regions maybe mixed. For example, a CH2 from a human IgG1 antibody could be joined with a CH3 region of a murine IgG2 antibody. Other such combinations analogous to this example would be apparent to those skilled in the art. It should be noted that many embodiments of the antigen-binding proteins described herein are often exemplified using the antigen-specific peptide S22; however, any of the antigen-specific peptides known to those of skill in the art, can be used to produce antigen-binding proteins using the protein scaffolds described herein.
In some embodiments, the described antigen-specific binding protein constructs may include amino acid resides that precede the antigen-specific peptide of the construct. Such residues may allow for increased protein stability, enhanced half-life, altered antigen binding, increased antigen affinity, or other such properties. In some aspects as many as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues may precede the antigen-specific peptide. In other embodiments methionine alone (M) may precede the antigen-specific peptide, such as provided in SEQ ID NO:3. In other embodiments the amino acids MA may precede the antigen-specific peptide, such as provided in residues 1 and 2 of SEQ ID NO:1. In one embodiments the amino acids MLCGGGG (residues 1-7 of SEQ ID NO: 4) may precede the antigen-specific peptide, such as provided in residues 1-7 of SEQ ID NO:4. In one embodiment the amino acids MLCGGGS (residues 1-7 of SEQ ID NO: 6) may precede the antigen-specific peptide, such as provided in residues 1-7 of SEQ ID NO:6. In one embodiments the amino acids MALMGGGG (residues 1-8 of SEQ ID NO: 7) may precede the antigen-specific peptide, such as provided in residues 1-8 of SEQ ID NO:7.
The described protein scaffolds and antigen-binding proteins can also include altered amino acid sequences that provide an enhanced protein function or reduce one or more undesirable property of the construct. These alterations may be achieved by a number of techniques known in the art, such as genetic engineering to alter the resulting amino acid sequence of the antigen-binding protein, chemical modification of the antigen-binding protein, or the additional functional peptides or motifs. In one embodiment, the described CH2 region, or Fc domain, may be altered to include one of more mutated amino acid residues. For example, residue 299 of the CH2 domain (or the amino acid corresponding to this residue in a related antigen-binding protein) may be modified to be a different amino acid. A modification of this sort may include mutating the native amino acid to be any other amino acid residue, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine. In one embodiment, residue 299 of the CH2 domain is changed from threonine to alanine. It will be understood by those skilled in the art that amino acid 299 of the CH2 region will not necessarily appear at position 299 in instances where a CH2 region is modified or incorporated to a larger scaffold or antigen-binding protein. Accordingly, in some embodiments, a residue of a protein-binding protein construct incorporating a CH2 region may be modified to encode a residue other than threonine at the amino acid corresponding to residue 299 of the CH2 region. For example, in one embodiment, a mutation of the residue corresponding to amino acid 299 of the CH2 region may occur at position 322 for an antigen-binding protein construct where the S22 peptide is linked to a CH2 region, since the addition of the S22 peptide adds N-terminal residues to the CH2 region. It should be noted that the numerical values noted for the described CH2 mutations are derived from the position of these amino acid residues in a full length IgG1 molecule and not their position in the context of the described scaffolds or antigen-binding proteins. To better exemplify this, the amino acid residue of the T322A mutation of SEQ ID NO:17 is underlined in Table 1. While this example is provide in the context of the CH2 peptide, other antigen specific peptides could alter the positioning of the CH2 residues in an analogous manner. Other scaffolds provided herein include LFcG7 (SEQ ID NO:16), LFcT322AG7 (SEQ ID NO:17), LFcT322AS7 (SEQ ID NO:18), and MFcG7 (SEQ ID NO:19) and MFcT322AG7 (SEQ ID NO:20). The amino acid sequences for each of these scaffolds shown in Table 1 includes a sequence of 12 “X” positions representing an antigen-specific peptide. While these placeholders are composed of 12 “X” positions, it should be understood that the antigen-specific peptide associated with the scaffold may contain a greater or lesser number of amino acid residues, such as SEQ ID NO:15 or any of SEQ ID NOs:24-44.







TABLE 1 







Described Sequences









SEQ ID NO.
Protein
Exemplary Amino Acid Sequence












1
S22-Fc
MAYCFPDEEGACYGGGGGSDKTHTCPPCPAPELLG




GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL




TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG




QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI




AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV




DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLE




HHHHHH


 


2
S22-FcT322A
MAYCFPDEEGACYGGGGGSDKTHTCPPCPAPELLG




GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE




VKFNWYVDGVEVHNAKTKPREEQYNSAYRVVSVL




TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG




QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI




AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV




DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSLE




HHHHHH


 


3
S22-FcT322A#4
MYCFPDEEGACYGGGGGYCFPDEEGACYGGGGGS




DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP




EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP




REEQYNSAYRVVSVLTVLHQDWLNGKEYKCKVSN




KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ




VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL




DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH




NHYTQKSLSLSLEHHHHHH


 


4
LS22FcG7
MLCGGGGYCFPDEEGACYGGGGGSDKTHTCPPCP




APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV




SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY




RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT




ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG




FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY




SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL




SLSLEHHHHHH


 


5
LS22FcT322AG7
MLCGGGGYCFPDEEGACYGGGGGSDKTHTCPPCP


 
also noted as
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV



S22-FcT322A#2
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSAY




RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT




ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG




FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY




SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL




SLSLEHHHHHH


 


6
LS22FcT322AS7
MLCGGGSYCFPDEEGACYGGGGGSDKTHTCPPCPA




PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS




HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSAYR




VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI




SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF




YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS




KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS




LSLEHHHHHH


 


7
MS22FcG7
MALMGGGGYCFPDEEGACYGGGGGSDKTHTCPPC




PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD




VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST




YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE




KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV




KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF




LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK




SLSLSLEHHHHHH


 


8
S22_s
GGGACCATGGCTTATTGCTTTCCGGATGAAGAAG




GTGCGTGCTACGGTGGTGGCGGCGGT


 


9
2S22
GGGACATATGTACTGTTTCCCGGACGAGGAGGGC




GCATGTTATG


 


10
2ndL
GAGGGCGCATGTTATGGCGGCGGTGGTGGCTATT




GCTTTCCGGAT


 


11
LS22
GGGACATATGTTGTGCGGCGGTGGTRGCTATTGC




TTTCCGGAT


 


12
MS22
GGGACCATGGCTTTAATGGGCGGTGGTGGCTATT




GCTTT


 


13
CH3rXho
GGGACTCGAGAGACAGGGAGAGGCTCTT


 


14
Fc
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP




EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP




REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN




KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ




VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL




DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH




NHYTQKSLSLSLE


 


15
S22 peptide
YCFPDEEGACY


 


16
LFcG7
MLCGGGGYCXXXXXXXXXXXXCYGGGGGSDKTH




TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC




VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ




YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP




APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT




CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD




GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH




YTQKSLSLSLEHHHHHH


 


17
LFcT322AG7
MLCGGGGYCXXXXXXXXXXXXCYGGGGGSDKTH




TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC




VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ




YNSAYRVVSVLTVLHQDWLNGKEYKCKVSNKALP




APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT




CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD




GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH




YTQKSLSLSLEHHHHHH


 


18
LFcT322AS7
MLCGGGSYCXXXXXXXXXXXXCYGGGGGSDKTH




TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC




VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ




YNSAYRVVSVLTVLHQDWLNGKEYKCKVSNKALP




APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT




CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD




GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH




YTQKSLSLSLEHHHHHH


 


19
MFcG7
MALMGGGGYCXXXXXXXXXXXXCYGGGGGSDK




THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV




TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE




EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA




LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS




LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS




DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN




HYTQKSLSLSLEHHHHHH


 


20
MFcT322AG7
MALMGGGGYCXXXXXXXXXXXXCYGGGGGSDK




THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV




TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE




EQYNSAYRVVSVLTVLHQDWLNGKEYKCKVSNKA




LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS




LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS




DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN




HYTQKSLSLSLEHHHHHH


 


21
Glycine-serine
GGGGGS



linker



 


22
CH2 domain
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED




PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS




VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK


 


23
CH3 domain
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP




SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS




LE


 


24
AHNP
FCDGFYACYMDV


 


25
AHNPY
YCDGFYACYMDV


 


26
AHNP-GG
FCDGFGGCYMDV


 


27
WP9QY
YCWSQYLCY


 


28
KP7-6
YCDEHFCY


 


29
OP3-4
YCEIEFCYLIR


 


30
reo3Y
FCIYSGSTCY


 


31
B2BPT
PCPINCTHSCVDLDDKGCPAEQRASPLTSI


 


32
B2APE
YCPIWKFPDEECY


 


33
EP1
YCGYSSTSYCFVMD


 


34
EP2
YCASRDYDYDGRCYFD


 


35
EP3
YCTRGYSSTSYCYAMD


 


36
EP4
FCMEESGGNYCY


 


37
EP5
YCALRGGVYWPCY


 


38
EP6
YCALTYYDYECFAY


 


39
CD4CDR3.AME
FCYICEVEDQCY



(82-89)



 


40
C3
ICVVQDWGHHRCT


 


41
WP5
YCFTASENHCY


 


42
E247-266
YCPALVTYNTDTFESMPNPEGRCY


 


43
CD3 gamma
QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFK




DGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQ




NKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFV




LAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPL




KDREDDQYSHLQGNQLRRN


 


44
CD3 epsilon
DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW




QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGY




YVCYPRGSKPEDANFYLYLRARVCENCMEMDVMS




VATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRG




AGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS




GLNQRRI









The scaffolds and antigen-binding proteins described herein can be made by recombinant processes and, therefore, may include amino acid sequences derived from more than one species (i.e. chimeric constructs) or may be engineered to have a human, or human-like, amino acid composition (i.e., a humanized construct). Accordingly, provided herein are vectors comprising polynucleotides capable of encoding the described scaffolds and antigen-binding proteins. The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus provided. The expression vector may contain one or more additional sequences such as, but not limited to, regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known to those of skill in the art. They include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors. The vectors described herein may be integrated into the host genome or maintained independently in the cell or nucleus.
Recombinant expression vectors contemplated to be within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′ nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated. Such vectors may be integrated into the host genome or maintained independently in the cell or nucleus.
The vectors described herein can be used to transform various cells with the genes encoding the disclosed scaffolds or antigen-binding proteins. For example, the vectors may be used to generate scaffold or antigen-binding protein-producing cells or cell lines. Thus, another aspect features host cells transformed with vectors comprising a nucleic acid sequence encoding a scaffold or antigen-binding protein, such as the scaffolds or antigen-binding proteins disclosed and exemplified herein. The host cells disclosed herein can be prokaryotic or eukaryotic cells. For example the host cell can be a bacteria. In one embodiment, the bacterial host cell is E. coli. Of course, the host cell can also be a mammalian cell, such as a Chinese hamster ovary (CHO) cell line. Numerous other such host cells, prokaryotic and eukaryotic, are known in the art and are considered to be within the scope of this disclosure.
Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the inventive methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like. Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.
Methods of using the described scaffolds, or antigen-binding proteins, are also provided. For example, antigen-binding proteins derived from the scaffolds disclosed herein may be used to treat or prevent disease in a subject. The described methods of treating or preventing disease can be used to administer compositions, having antigen-binding proteins derived from the described scaffolds, to a subject in need of such treatment. In some embodiments, the described methods of treatment can be used to treat a subject having cancer. In some embodiments the methods of treatment described herein can be used to treat a subject having cancer that is susceptible to treatment with a HER2 binding protein, such as a HER2-specific antibody. In some embodiments the methods of treatment described herein can be used to treat a subject having cancer that is susceptible to treatment by preventing HER3 signaling. In some embodiments the methods of treatment described herein can be used to treat a subject having cancer that is susceptible to treatment by disrupting dimerization of HER3. In some embodiments the methods of treatment described herein can be used to treat a subject having cancer that is susceptible to treatment by disrupting dimerization of HER2 and HER3. In some embodiments the methods of treatment described herein can be used to treat a subject having cancer that is known to be resistant to treatment with a HER2-specific antibody. In some embodiments the methods of treatment described herein can be used to treat a subject having cancer that is known to be resistant to treatment with a EGFR-specific antibody. Also disclosed are methods for detecting an antigen of interest using antigen-binding proteins derived from the scaffolds disclosed herein. Such methods are applicable to antigen detection in a subject, in a sample obtained from a subject, or in vitro. The methods described herein can be particularly applicable to treating or preventing diseases associated with HER2 such as administering to a subject an antigen-binding protein capable of interacting with HER2, as disclosed herein, and a pharmaceutically acceptable carrier. Methods described herein can also be used to treat or prevent diseases associated with the EGFR, such as administering to a subject an antigen-binding protein capable of interacting with EGFR, as disclosed herein, and a pharmaceutically acceptable carrier. In addition, methods described herein can also be used to treat or prevent diseases associated with the HER3, such as administering to a subject an antigen-binding protein capable of interacting with HER3, as disclosed herein, and a pharmaceutically acceptable carrier
Alternatively, antigen-binding proteins derived from the scaffolds disclosed herein may be used to detect disease-causing agents or disease associated proteins or metabolites in a subject or a sample obtained from a subject, which in turn can allow for a diagnosis. The methods described herein can be particularly applicable to detecting or otherwise assessing the expression of the HER2, EGFR, or HER3 in a subject. For example, one could inject a subject with a detectably labeled embodiment of an antigen-binding protein capable of binding HER2, EGFR, or HER3, as described herein, and detect the localization and/or intensity of the signal in the subject. Alternatively, one could expose a sample containing HER2, EGFR, or HER3 to a described antigen-binding protein and detecting binding of the antigen-binding protein to the sample.
In some embodiments, the disclosed antigen-binding proteins are conjugated to one or more chemotherapeutic agents such as, but not limited to radionuclides, toxins, and cytotoxic and cytostatic agents. In other embodiments the antigen-binding proteins are used in combination with one or more chemotherapeutic agents. The antigen-binding proteins described herein may be used alone or with (e.g., coadministered or conjugated to) a biomolecule or chemotherapeutic agent such as a cytotoxic or cytostatic agent. In some embodiments, the chemotherapeutic agent is a radionuclide, including, but not limited to lead-212, bismuth-212, astatine-211, iodine-131, scandium-47, rhenium-186, rhenium-188, yttrium-90, iodine-123, iodine-124, iodine-125, bromine-77, indium-111, and fissionable nuclides such as boron-10 or an actinide. In other embodiments, the chemotherapeutic agent is a toxin or cytotoxic drug, pokeweed antiviral protein, abrin, ricin and each of their A chains, momordin, saporin, bryodin 1, bouganin, gelonin, Diphtheria toxin, Pseudomonas exotoxin, Shiga toxin, calicheamicin, maytansinoid, altretamine, actinomycin D, plicamycin, puromycin, gramicidin D, doxorubicin, colchicine, cytochalasin B, cyclophosphamide, emetine, maytansine, amsacrine, cisplastin, etoposide, etoposide orthoquinone, teniposide, daunorubicin, gemcitabine, doxorubicin, mitoxantraone, bisanthrene, Bleomycin, methotrexate, pemetrexed, cisplatinum, vindesine, adriamycin, vincristine, vinblastine, BCNU, taxol, tarceva, avastin, mitomycin, 5-fluorouracil, cyclophosphamide, certain cytokines such as TNF-alpha and TNF-beta, and the like. Methods of conjugation of scaffolds or antigen-binding proteins to such agents are known in the literature.
Described herein are compositions comprising at least one disclosed scaffold or antigen-binding protein and a pharmaceutically acceptable carrier. The compositions can be formulated as any of various preparations that are known and suitable in the art, including those described and exemplified herein. In some embodiments, the compositions are aqueous formulations. Aqueous solutions can be prepared by admixing the antigen-binding proteins in water or suitable physiologic buffer, and optionally adding suitable colorants, flavors, preservatives, stabilizing and thickening agents and the like as desired. Aqueous suspensions can also be made by dispersing the antigen-binding proteins in water or physiologic buffer with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Also included are liquid formulations and solid form preparations which are intended to be converted, shortly before use, to liquid preparations. Such liquids include solutions, suspensions, syrups, slurries, and emulsions. Liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats or oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). These preparations may contain, in addition to the active agent, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The compositions may be in powder or lyophilized form for constitution with a suitable vehicle such as sterile water, physiological buffer, saline solution, or alcohol, before use.
The compositions can be formulated for injection into a subject. For injection, the compositions described can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Injection formulations may also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.
The compositions can be formulated in sustained release vehicles or depot preparations. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.
The antigen-binding proteins described herein may be administered orally in any acceptable dosage form such as capsules, tablets, aqueous suspensions, solutions or the like. The antigen-binding proteins may also be administered parenterally including but not limited to: subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intranasal, topically, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Generally, the antigen-binding proteins will be intravenously or intraperitoneally, for example, by injection.
The subject can be any animal, and preferably is a mammal such as a mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, donkey, cow, horse, pig, and the like. In some instances the subject is a human. In some embodiments, subjects can be administered at least one antigen-binding protein in a daily dose range of about 0.01 μg to about 500 mg of antigen-binding protein per kg of the weight of the subject. The dose administered to the subject can also be measured in terms of total amount of the at least one antigen-binding protein administered per day. In some embodiments, a subject is administered about 5 to about 5000 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 10 milligrams of at least one a antigen-binding protein per day. In some embodiments, a subject is administered up to about 100 milligrams of at least antigen-binding protein per day. In some embodiments, a subject is administered up to about 250 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 500 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 750 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 1000 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 1500 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 2000 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 2500 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 3000 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 3500 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 4000 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 4500 milligrams of at least one antigen-binding protein per day. In some embodiments, a subject is administered up to about 5000 milligrams of at least one antigen-binding protein per day. In some embodiments, the antigen-binding protein is administered to a subject weekly or bi-weekly.
For effective treatment, one skilled in the art may recommend a dosage schedule and dosage amount adequate for the subject being treated. It may be preferred that dosing occur one to four or more times daily for as long as needed. The dosing may occur less frequently if the compositions are formulated in sustained delivery vehicles. The dosage schedule may also vary depending on the active drug concentration, which may depend on the needs of the subject.
There are several embodiments that follow from the description provided above, particularly those relating to antigen-binding proteins. In one embodiment, an antigen-binding protein can have at least one protein scaffold and at least one antigen-specific peptide. The antigen-binding proteins described above could be composed from a protein scaffold such as any one, or more, of the protein scaffolds exemplified by the amino acid sequences shown in Table 1. In some embodiments, the protein scaffold will have only the amino acid sequences that are between the glycine-serine linker (GGGGGS) (SEQ ID NO: 21) and the histidine tag (HHHHHH) (SEQ ID NO: 45) of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, while in other embodiments the protein scaffold can further include the amino acid segment preceding the antigen-specific peptide for SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, in addition to other amino acid sequences. In addition, the described antigen-binding proteins can incorporate any number of antigen-specific peptides, such as the antigen-specific peptide of SEQ ID NO:15. Furthermore, the described antigen-binding proteins can incorporate one or more linkers, made of at least one glycine residue, that connect the components of the protein. In a particular embodiment, the linker described above can have an amino acid sequence of, or similar to, that of SEQ ID NO:20. In some embodiments, the linker will have only the amino acid sequence of SEQ ID NO:20, while in other embodiments a linker can include SEQ ID NO:20 in addition to other amino acid sequences. In other embodiments, any of the antigen-binding proteins described herein can have an epitope tag, a fluorophore, a radio isotope, or an enzyme. In a preferred embodiment, the embodied antigen-binding protein has an epitope tag that is a poly-histidine tag.
The described antigen-binding proteins can be encoded by a variety of polynucleotides capable of encoding the amino acid sequences provided herein. These polynucleotides can also be incorporated into vectors useful for the maintenance, replication, and/or expression of the polynucleotides encoding the described antigen-binding proteins or the described portions thereof. The vectors described above can be used to engineer cells to express the antigen-binding proteins or the described portions thereof encoded by the polynucleotides disclosed herein.
Also provided herein are compositions that include at least one of the antigen-binding proteins described herein and a pharmaceutically acceptable carrier. Such compositions are useful in the methods provided herein for treating or preventing disease in a subject. In one embodiment, the described methods of treatment include administering a therapeutic amount of a one or more of the described antigen-binding proteins to a subject in need of such treatment. Similarly, in one embodiment, the described methods of preventing disease in a subject include administering a therapeutic amount of a one or more of the described antigen-binding proteins to a subject in need thereof. A preferred method of treating or preventing a disease relate to a disease associated with HER2, EGFR, or HER3 in a subject, where the treatment or prevention includes administering to the subject a composition including an antigen-binding protein described herein. Also provided are methods of detecting an antigen of interest in a subject that include administering an antigen-binding protein provided herein to a subject and detecting binding of the antigen-binding protein to an antigen of interest. A preferred method of detection involves using at least one of the antigen-binding proteins described to detect HER2 by exposing a sample containing HER2 to an antigen-binding protein capable of binding HER2 and detecting binding of at least one antigen-binding protein to the sample. Another preferred method of detection involves using at least one of the antigen-binding proteins described to detect EGFR by exposing a sample containing EGFR to an antigen-binding protein capable of binding EGFR and detecting binding of at least one antigen-binding protein to the sample. Another preferred method of detection involves using at least one of the antigen-binding proteins described to detect HER3 by exposing a sample containing HER3 to an antigen-binding protein capable of binding HER3 and detecting binding of at least one antigen-binding protein to the sample.
Also provided herein are compositions that include the antigen-binding proteins described herein and a pharmaceutically acceptable carrier. Such compositions are useful in the methods provided herein for treating or preventing disease in a subject. In one embodiment, the described methods of treatment include administering a therapeutic amount of a one or more of the described antigen-binding proteins to a subject in need of such treatment. Similarly, in one embodiment, the described methods of preventing disease in a subject include administering a therapeutic amount of a one or more of the described antigen-binding proteins to a subject in need thereof. A preferred method of treating or preventing a disease relate to a disease, such as cancer, associated with HER2, EGFR, or HER3 in a subject, where the treatment or prevention includes administering to the subject a composition including a antigen-binding protein described herein. Also provided are methods of detecting an antigen of interest in a subject that include administering an antigen-binding protein provided herein to a subject and detecting binding of the antigen-binding protein to an antigen of interest. A preferred method of detection involves using at least one of the antigen-binding proteins described to detect HER2, EGFR, or HER3 by exposing a sample containing one of these receptors to an antigen-binding protein and detecting binding of at least one antigen-binding protein to the sample.
The following examples are provided to further describe some of the embodiments disclosed herein. They are intended to illustrate, not to limit, the disclosed embodiments.
EXAMPLE I
Design and Expression of S22-Fc Constructs
A recombinant expression strategy was developed in order to embed the S22 peptide into an intact Fc antibody segment, having both CH2 and CH3 domains of IgG1 (FIG. 1). Since S22 is a very short peptide, the cDNA for the S22-Fc chimera was produced by PCR using 5′ primers containing the S22 sequence. A plasmid containing human IgG1 cDNA clone (ATCC no. 9898675) was used as the PCR template. Proper restriction sites were designed at 5′ and 3′ ends so the resulting PCR products could be cloned into the E. coli expression vector pET21d (Novagen). E. coli expression was selected because it allows for the rapid production of recombinant proteins. In E. coli, the S22-Fc fusion proteins are predominantly expressed in inclusion bodies as insoluble proteins, but some of it is also present in the soluble fraction, especially when the bacteria are cultured at 16° C. (FIG. 2). Soluble fusion proteins were purified on Ni-sepharose columns according to manufacturer's instructions (GE Healthcare).
FACS analysis was used to detect binding of S22-Fc to HER2-expressing cell lines. Purified S22-Fc was used in FACS experiments to stain cells with different expression levels of HER2 and EGFR. Initially, 1×105 cells were incubated with 10 μg S22-Fc for 30 minutes on ice. The cells were then washed and incubated with his-tag-specific rabbit antisera and secondary Alexa488-conjugated anti-rabbit antibodies, used sequentially. As a negative control, parallel cell preparations were exposed to only his-tag-specific rabbit antisera and secondary antibodies.
FACS analysis showed that S22-Fc bound to T6-17 cells (a mouse fibroblast engineered to express human HER2) and NE91 cells (A mouse fibroblast cell line engineered to express human EGFR); however, MCF7 cells (a human breast cancer cell line negative for HER2 and minimally positive for EGFR) showed minimal binding (FIG. 3). These data indicate that embedded Fc forms that have S22 CDR binding units and intact Fc regions can be generated and are functional.
EXAMPLE II
Modification of the CH2 Domain of S22-Fc Improves Binding
Sazinsky et al. (Proc. Natl. Acad. Sci. U.S.A., 105(51):20167-72 (2008)), described a T299A mutation near the glycosylation site (N297) in the CH2 domain that dramatically improved the ability of a glycosylated Fc fragment to bind to Fc receptors. This mutation was introduced to the S22-Fc construct in order to improve its binding with Fc receptors. The resulting construct was named S22FcT322A construct (FIG. 4). Unexpectedly, the introduction of this mutation resulted in reduced HER2 (on T6-17 cells) and EGFR (on NE91 cells) binding for S22FcT322A. However, another construct, S22FcT322A#2, which contains the T299A mutation but accidentally expressed several additional amino acids at the N-terminal, possessed better binding than S22-Fc to T6-17 and NE91 cells (FIG. 4). The observed improved binding is expected to be a consequence of improved stability of the construct. No constructs stained the control cell line MM3MG (a murine fibroblast cell line). In addition, the S22-Fc and S22FcT322A#2 constructs also showed some binding to MCF7 cells, which does not overexpress HER2 and EGFR, but is known to express HER3.
Based on these initial findings, additional modifications were made to the S22-Fc construct to produced other derivative fusion proteins: LS22FcG7; LS22FcT322AG7 and LS22FcT322AS7 As shown in FIG. 5, LS22FcG7, which has the wild type Fc fragment, had the highest affinity to T6-17 cells, while LS22FcT322AG7 also bound cells better than S22Fc. Another construct, MS22FcT322AG7, appeared to exhibit even greater binding as observed by FACS analysis (data not shown).
EXAMPLE III
Inhibition of Tumor Growth
The LS22FcT322AG7 antigen-binding protein was tested to assess its ability to inhibit tumor growth in vivo. In these experiments LS22FcT322AG7 was tested against the anti-HER2 antibody 4D5 (positive control) for the ability to inhibit tumors induced by the T6-17 cell line that expresses human HER2/neu. Initially, 5×104 T6-17 cells were subcutaneously inoculated into athymic nude mice. Mice carrying a tumor received either LS22FcT322AG7, the 4D5 antibody, or control buffer at the dose of 10 mg/kg, three times per week via intraperitoneal (i.p.) injection. As shown in FIG. 6, both LS22FcT322AG7 and the 4D5 antibody inhibited tumor growth.
Since S22-Fc antigen-binding proteins also bind to EGFR, experiments were conducted to determine if some of these constructs could also inhibit tumor xenografts induced by the A431 cell line, which overexpresses human EGFR. For these experiments, 1×106 A431 cells were subcutaneously inoculated into athymic nude mice. Mice carrying tumor received LS22FcT322AG7, LS22Fc or control buffer at the dose of 10 mg/kg, three times per week via i.p. injection. As shown in FIG. 7, both LS22FcT322AG7 and LS22Fc demonstrated activity in the inhibition of A431 tumor growth.
EXAMPLE IV
LS22FcT322AG7 Blocks Binding of HER2-Specific Antibodies
The antigen-binding protein LS22FcT322AG7 contains the S22 loop from HER2, which is located within subdomain IV of the receptor's extracellular domain, where the anti-HER2 antibody 4D5 (trastuzumab) binds (Cho et al., Nature 421:756-760 (2003)). Given these characteristics, experiments were conducted to determine whether antigen-binding protein LS22FcT322AG7 could bind to antibodies that are specific for HER2 and also whether antigen-binding protein LS22FcT322AG7 could block binding of antibodies specific for HER2. For these studies, Biacore experiments were carried out using purified LS22FcT322A immobilized to sensor chip following the standard amine coupling procedure according to the manufacturer's instructions. Then variable concentrations (5 μM, 15 μM, 45 μM) of HER2-specific antibody 7.16.4 (Drebin, J. A., et al., Nature 312:545-548 (1984)) were run over the chip surface to determine binding affinity. As shown in FIG. 9A, binding kinetics were observed for each concentration of antibody 7.16.4. The binding affinity of antibody 7.16.4 for LS22FcT322AG7 was determined to be a KD of 1.4 μM. FACS experiments were then carried out to determine whether the antigen-binding protein LS22FcT322AG7 could inhibit binding of the murine 4D5 antibody (m4D5) to HER2/neu receptors on T6-17 cells in a dose-dependent manner. For these experiments human HER2-expressing T6-17 cells were incubated with antibody m4D5 in the presence of different concentrations (0 μg, 10 μg, or 50 μg) of LS22FcT322AG7. FITC-labeled anti-mouse secondary antibody was used to detect m4D5 bound to cells. As shown in FIG. 9B, reduced binding of m4D5 was observed in the presence of LS22FcT322AG7 and occurred in a dose-dependent manner. These data indicate that LS22FcT322AG7 could be used to target, and disrupt, HER2 interactions that occur through domain IV of the receptor.
EXAMPLE V
LS22FcT322AG7 is Effective Against Tumor Cells that Resist Treatment with Anti-EGFR and Anti-HER2 Antibodies
The tumor cell line HCl has been previously characterized as being resistant to treatment with cetuximab, an EGFR-specific antibody that is FDA-approved for treating cancer (Wheeler et al., Oncogene 27(28):3944-56 (2008)). The cell line has also been shown to have increased levels of heterodimeric ErbB receptors (Id.). Experiments were conducted to assess whether HCl cells were sensitive to treatment with LS22FcT322AG7. Initial experiments showed that the presence of LS22FcT322AG7 could prevent epidermal growth factor (EGF) and heregulin (HGR)-mediated HER3 activation in cultured HCl cells (FIGS. 10A and 10B). Furthermore, treatment with 10 mg/kg per week of LS22FcT322AG7 was shown to reduce the growth of HCl xenographs implanted subcutaneously into athymic mice (FIG. 10C).
Further studies were conducted to assess the ability of LS22FcT322AG7 to inhibit the growth of xenographs in mice from the human breast cancer cell line TB129, which was derived from patients who did not respond to treatment with trastuzumab (anti-HER2 antibody). These cells express HER2, but are resistant to trastuzumab in vitro and in vivo. For these experiments tumor cell line TB129 was implanted into nude mice subcutaneously. Nine days after implantation, mice started to receive intraperitoneal treatment with 10 mg/kg of LS22FcT322AG7 three times per week. As shown in FIG. 11, tumor growth was inhibited in mice treated with LS22FcT322AG7 versus mice receiving no treatment (control)).

## Claims

1. A recombinant antigen-binding protein comprising the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7.

2. A recombinant antigen-binding protein comprising the protein scaffold of SEQ ID NO:14, 16, 17, 18, 19, or 20.

3. The recombinant antigen-binding protein of claim 2, further comprising an antigen-specific peptide of SEQ ID NO:15, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

4. The recombinant antigen-binding protein of claim 2, further comprising an epitope tag, a fluorophore, a radio isotope, or an enzyme.

5. The recombinant antigen-binding protein of claim 4, wherein said epitope tag is a poly-histidine tag.

6. A composition comprising the recombinant antigen-binding protein of claim 1 and a pharmaceutically acceptable carrier.

7. A composition comprising the recombinant antigen-binding protein of claim 2 and a pharmaceutically acceptable carrier.