WO2014107165A1

WO2014107165A1 - Fully human antibodies against human receptor igf-1r

Info

Publication number: WO2014107165A1
Application number: PCT/US2013/020424
Authority: WO
Inventors: Tajib Mirzabekov; Ilya ALEXANDROV; Khikmet SADYKOV; Anton CHESTUKHIN; Roman MIKHAILOV; Alexey REPIK; Vasily IGNATIEV; Mikhail Samsonov; Yan Lavrovsky
Original assignee: R-PHARM, CJSC (Closed Joint Stock Company)
Priority date: 2013-01-05
Filing date: 2013-01-05
Publication date: 2014-07-10

Abstract

Therapeutic antibodies are described that can be used for treating or prevention of diseases associated with modulation of activity of human IGF-1R. In certain aspects, the disclosed invention is based upon discovering antibodies that are capable of binding to human IGF-1R. The disclosed antibodies are capable of blocking the interactions between IGF-1R and its ligand IGF-1 and are capable of attenuating cell surface presentation of IGF-1R. The disclosed antibodies could be administered to humans without additional modifications such as a "humanization" process. The antibodies could also be used as diagnostics and for research purposes. The described monoclonal antibodies are applicable for IGF-1R /IGF-1 signaling cascade targeting in drug development for cancer and immunological disorders.

Description

Fully Human Antibodies Against Human Receptor IGF-IR.

FIELD

The invention generally relates to the field of molecular biology, immunology, autoimmune and inflammatory diseases and oncology. More specifically, the invention relates to antibodies that bind to human receptor IGF-IR.

BACKGROUND

The Insulin-Like Growth Factor (IGF) pathway plays a major role in cancer proliferation, survival and resistance. Therefore this pathway is the target of many investigational

pharmacologic agents. Insulin-Like Growth Factors IGF-1 and -II are potent cancer growth stimulators. These circulating ligands signal through IGF-IR receptor, insulin receptor (IR) isoform A (IR-A), insulin receptor isoform B (IR-B), IGF-2R receptor and IGF binding proteins (IGFBP) 1 through 6. The IGF-IR has been the main target of therapy directed against the IGF signaling system, as it is a major transducer of IGF signaling leading to proliferation.

IGF-IR is expressed on the cell surface as a heterotetramer composed of two extracellular a chains and two membrane-spanning b chains in a disulfide-linked b-a-a-b. On binding to ligand, IGF- IR undergoes conformational changes and autophosphorylation. Ultimately, through subsequent

phosphorylation of intracellular substrates, the MAPK and PI3K/Akt pathways are activated. Activation of these pathways has been shown to lead to cell proliferation and resistance to apoptosis. IGF- IR belongs to the IR family that includes the IR, IGF- IR (homodimer), IGF-1R/IR (hybrid receptor) and IGF-2R. IGF- lR/IR hybrids have biological activity similar to IGF-IR homoreceptors, preferentially binding and signaling with IGF ligands, rather than with insulin. While IGF-I and -II are abundant in the serum of adults, there are several proteins that limit their bioavailability, and thus ability to activate IGF- IR. Six IGFBP and non-signaling IGF-II binding IGF-2R bind circulating IGF-I/II to limit their bioavailability down to ~ 2%. High circulating levels of IGF-I and increased expression of the IGF- IR are associated with an increased risk of developing breast, prostate, colon, lung, thyroid and other cancers thus, the inhibition strategies may have clinical relevance in a large number of tumors.

There are several IGF-IR blocking monoclonal antibodies (MABs) in development. One such antibody is cixutumumab (CIX), a fully human IgGl that binds IGF-IR with high affinity and blocks interaction between IGF-IR and its ligands, IGF-1 and -II, and induces internalization and degradation of IGF-IR. IGF-IR is a member of the receptor tyrosine kinase family. Upon binding IGF-1 receptor IGF-IR autophosphorylates tyrosine residues, thereby initiating signaling. Binding of a blocking MAB results in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2.

Due to the fact that receptor pairs within the IGF system are covalently bound, the anti IGF-IR MABs have the ability to not only block IGF-IR homodimers, but also hybrid IGF-IR/IR receptors in tumor cells. CIX binds to the IGF-IR with high affinity (Kd = 40 pM) and causes subsequent

internalization and degradation of the receptor. CIX has been shown to indirectly inhibit both the ERK- MAPK as well as the PI3 K- AKT-mTOR pathway through IGF-IR receptor blockade.

Biological agents have become a major focus in novel therapeutic development for cancer because of their potential for highly specific molecular and cellular targeting combined with their typically low toxicity compared with small-molecule drugs. The rationale for using multiple agents directed at more than a single target stems from efforts to minimize the emergence of drug resistance and to exploit the effectiveness of combined therapies. Development of antibodies capable of altering the signaling characteristics of IGF-IR began more than 20 years ago, when the receptor's potential importance in cancer pathogenesis became clear.

Yamashita et al. (1986) reported that the monoclonal antibody aIR3 could block the effects of IGF-1 on growth hormone production in human pituitary adenoma cells. Subsequently, Li et al. (1993) described another monoclonal antibody, designated 1H7, that could block the ligation and activation of IGF-IR. In the years that followed, a number of anti-IGF-lR antibodies have been developed. Doern et al. (2009) recently described an extensive panel of antibodies that can inhibit IGF-IR activation and the downstream events associated with its signaling. They screened and categorized these antibodies and divided them into four groups on the basis of their abilities to block receptor ligation by both IGF- 1 and IGF-II. They found antibodies that could allosterically block either IGF- 1 or IGF-II, allosterically block both, or competitively block both IGF-1 and IGF-II binding to IGF-IR. Their epitope mapping studies used three separate constructs, including human IGF-IR 1-903, mouse 1-904, and human IGF- IR 1-462, which contains the three N-terminal domains, including LI, the cysteine -rich region, and L2. Using a purified IGF- IR library including 64 mutations, they found that the epitopes recognized by antibodies belonging to all four categories bound overlapping surfaces of the cysteine-rich repeat and L2 domains. BIIB4 blocks IGF-1 and IGF-II competitively, whereas BIIB5 blocks both ligands through an allosteric mechanism. Furthermore, the authors found that binding of IGF- 1 and these antibodies resulted in conformation changes in IGF-IR (Doern et al., 2009). These detailed studies may prove invaluable in dissecting the properties of individual antibodies that render optimal results in subsequent clinical trials.

At least eight different monoclonal antibodies directed at the IGF-IR are currently under development or are being assessed in clinical trials. Each exhibits unique characteristics and has been developed using different strategies. These have been well reviewed. A spectrum of receptor internalization and blocking activities found among these antibodies should thus put these findings into a perspective of clinical efficacy.

Further discussion of therapeutic utility of anti-IGF-lR antibodies can be found in the references cited below:

Feng Y, and Dimitrov, DS 2008 Monoclonal antibodies against components of the IGF system for cancer treatment. Curr Opin Drug Discov Devel 1 1 : 178-185.

Doern A, et al. 2009 Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-b locking properties: implications for mechanism of action in vivo. J Biol Chem 284: 10254-10267

Cohen et al., 2005 Combination therapy enhances the inhibition of tumor growth with the fully human anti-type 1 insulin-like growth factor receptor monoclonal antibody CP-751 ,871. Clin Cancer Res 1 1 :2063-2073.

Pollak et al., 2007. Pharmacodynamic properties of the anti-IGF-IR monoclonal antibody CP-751 ,871 in cancer patients. J Clin Oncol 25 (Suppl 18S):3587.

Abroun et al., 2004. Receptor synergy of interleukin-6 (IL-6) and insulin-like growth factor-I in myeloma cells that highly express IL-6 receptor a [published erratum appears in Blood 103 :2891 , 2004]. Blood 103 :2291-2298.

Olmos et al., 2007. Safety tolerability and preliminary efficacy of the anti-IGF-IR monoclonal antibody CP-751 ,871 in patients with sarcomas and adrenocortical tumors, in 19th Annual AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 22-26 Oct 2007; San Francisco, CA. Poster A63.

Burtrum et al, 2003. A fully human monoclonal antibody to the insulin-like growth factor-I receptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo. Cancer Res 63 :8912-8921.

Rowinsky et al., 2007. IMC-A12, a human IgGl monoclonal antibody to the insulin-like growth factor-I receptor. Clin Cancer Res 13 :5549s-5555s.

Hariharan et al., 2007. BIIB022, a fully human nonglycosylated γ4Ρ antibody targeting IGF- 1R for cancer therapy, in 19th Annual AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 22-26 Oct 2007; San Francisco, CA. Poster B210. Goetsch et al, 2005. A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts. Int J Cancer 113:316-328.

Schnitzer et al., 2006. Characterization of a recombinant, fully human monoclonal antibody directed against the human insulin- like growth factor- 1 receptor. Eur J Cancer 42 (Suppl): 66-67.

Rondon et al., 2007. A phase I study of q3W R1507, a human monoclonal antibody IGF- IR (insulin- like growth factor receptor) antagonist in patients with advanced solid tumors, in 19th Annual AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 22-26 Oct 2007; San Francisco, CA. Poster A77.

Maloney et al., 2003. An anti-insulin-like growth factor-I receptor antibody that is a potent inhibitor of cancer cell proliferation. Cancer Res 63:5073-5083.

Descamps et al, 2006. CD45neg but not CD45pos human myeloma cells are sensitive to the inhibition of IGF-1 signaling by a murine anti-IGF-lR monoclonal antibody, mAVE1642. J Immunol 177:4218-4223.

Descamps et al., 2009. A humanised anti-IGF-lR monoclonal antibody (AVE1642) enhances

Bortezomib-induced apoptosis in myeloma cells lacking CD45. Br J Cancer 100:366-369.

Pandini et al., 1999. Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling. Clin Cancer Res 5: 1935-1944.

Sachdev et al, 2006. Down-regulation of insulin receptor by antibodies against the type I insulin-like growth factor receptor: implications for anti-insulin-like growth factor therapy in breast cancer. Cancer Res 66:2391-2402.

Pfister DG, Johnson DH, Azzoli CG, Sause W, Smith TJ, Baker S Jr, Olak J, Stover D,

Strawn JR, Turrisi AT, Somerfield MR: American Society of Clinical Oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol 2004, 22:330-353.

Choi YJ, Rho JK, Jeon BS, Choi SJ, Park SC, Lee SS, Kim HR, Kim CH, Lee JC 2010

Combined inhibition of IGFR enhances the effects of gefitinib in HI 650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors. Cancer Chemother

Pharmacol. 66(2): 381-388. Burtrum D, Zhu Z, Lu D, et al. A Fully human monoclonal antibody to the insulin-like- growth factor I receptor blocks ligand-dependent signaling and inhibits tumor growth in vivo. Cancer Res 2003;63:8912-8921.

Wang Z, Chakravarty G, Kim S, et al. Growth-inhibitory effects of human anti-insulin-like growth factor I receptor antibody (A 12) in an orthotopic nude mouse model of anaplastic thyroid carcinoma. Clin Cancer Res 2006;12:4755-4765.

Wu KD, Zhou L, Burtrum D, et al. Antibody targeting of the insulin-like-growth factor I receptor enhances the anti-tumor response of multiple myeloma to chemotherapy through inhibition of tumor proliferation and angiogenesis. Cancer Immunol Immunother 2007;56:343- 357.

Wu JD, Odman A, Higgins LM, et al. In vivo effects of the human type I insulin-like growth factor receptor antibody A12 on androgen-dependent and androgen-independent xenograft human prostate tumors. Clin Cancer Res 2005; 11 :3065-3074.

Anti-insulin- like growth factor-I receptor (IGF-1R) monoclonal antibody investigator's brochure. Imclone Systems, Inc; 2006.

Bertrand FE, Steelman LS, Chappell WH, et al. Synergy between an IGF-1R antibody and Raf/MEK/ ERK and PI3K/Akt/mTOR pathway inhibitors in suppressing IGF-1R mediated growth in hematopoietic cells. Leukemia 2006;20: 1254-1260.

Tonra JR, Deevi DS, Corcoran E, et al. Combined antibody mediated inhibition of IGF-1R, EGFR, VEGFR2 for more consistent and greater anti-tumor effects. Eur J Cancer 2006;4:207.

Wu JD, Haugk K, Coleman I, et al. Combine in vivo effect of A12, a type 1 insulin-like growth factor receptor antibody, and docetaxel against prostate cancer tumors. Clin Cancer Res 2006;12(20 Pt 1): 6153-6160.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery of antibodies that specifically bind to human IGF-1R blocking the IGF/IGF- 1R signaling. Series of antibodies were isolated from antibody libraries that contained fully human antibody frames. The CDRs (Complementarity Determining Regions) of IGF-1R -specific antibodies differ from each other, consistent with the design of the antibody libraries used. In these libraries, CDRl and CDR2 of the antibody heavy chain were randomized to provide limited number of variation and CDR3 of the heavy chain was randomized to include all possible amino acid combinations. For the light chain, CDRl and CDR2 were invariant and CDR3 was randomized completely. Since the frameworks for both heavy and light chain of antibodies were corresponding to human sequences, the antibodies described herein could be administered to humans without additional modifications such as a "humanization" process. The antibodies could also be used as diagnostics and for research purposes.

The monoclonal antibodies disclosed herein are applicable for IGF-1R /IGF-1 signaling cascade targeting in drug development for cancer and immunological disorders, such as Crohn's and RA, and others.

In some aspects the invention provides for an isolated antibody (IR3), or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain of SEQ ID NO. 61, and an immunoglobulin heavy chain of SEQ ID NO. 55. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody (IR4), or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain of SEQ ID NO. 62, and an immunoglobulin heavy chain of SEQ ID NO. 56. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody (IR4A), or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain of SEQ ID NO. 63, and an immunoglobulin heavy chain of SEQ ID NO. 57. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody (IR8), or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain of SEQ ID NO. 64, and an immunoglobulin heavy chain of SEQ ID NO. 58. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody (IR10), or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain of SEQ ID NO. 65, and an immunoglobulin heavy chain of SEQ ID NO. 59. The antibody can be a monoclonal antibody. In some aspects the invention provides for an isolated antibody (IR24), or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain of SEQ ID NO. 66, and an immunoglobulin heavy chain of SEQ ID NO. 60. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 45. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ ID NO. 13, a CDRH2 comprising the sequence of SEQ ID NO. 14, and a CDRH3 comprising the sequence of SEQ ID NO. 15. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 46. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ ID NO. 16, a CDRH2 comprising the sequence of SEQ ID NO. 17, and a CDRH3 comprising the sequence of SEQ ID NO. 18. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 47. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRH1 comprising the sequence of SEQ ID NO. 19, a CDRH2 comprising the sequence of SEQ ID NO. 20, and a CDRH3 comprising the sequence of SEQ ID NO. 21. The CDR sequences of the antibody can be interposed between human or humanized framework sequences. In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 48. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 22, a CDRH2 comprising the sequence of SEQ ID NO. 23, and a CDRH3 comprising the sequence of SEQ ID NO. 24. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 49. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 25, a CDRH2 comprising the sequence of SEQ ID NO. 26, and a CDRH3 comprising the sequence of SEQ ID NO. 27. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising a CDRLl comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 50. The antibody further comprises an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 28, a CDRH2 comprising the sequence of SEQ ID NO. 29, and a CDRH3 comprising the sequence of SEQ ID NO. 30. The CDR sequences of the antibody can be interposed between human or humanized framework sequences.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 31 , and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 01. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 33, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 03. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 35, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 05. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 37, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 07. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 39, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 09. The antibody can be a monoclonal antibody.

In some aspects the invention provides for an isolated antibody, or an antigen binding fragment of the antibody, that binds human IGF-1R receptor. The antibody comprises an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 41, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 11. The antibody can be a monoclonal antibody.

These and other aspects and advantages of the invention described herein will become apparent upon consideration of the Figures and detailed description of antibody properties below. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings and descriptions provide complete understanding of the invention:

Figure 1 schematically shows representation of a typical antibody. Gray areas on the scheme depict Constant Regions of both antibody light and heavy chains, black areas depict Variable Regions of the antibody light chain, white areas depict Variable Regions of the antibody heavy chain. CHI, CH2 and CH3 - constant regions 1, 2 and 3of the antibody heavy chain respectively. VH - Variable Regions of the antibody heavy chain; VL - Variable Regions of the antibody light chain; CL - Constant Region of the antibody light chain. Links between heavy and light chains and between two heavy chains depict intermolecular disulfide bridges;

Figure 2 schematically shows the amino acid sequences defining a complete immunoglobulin Heavy Chain Variable Region of the antibodies assigned as IR3, IR4, IR4A, IR8, IR10, and IR24. The amino acid sequences are aligned relative to each other where the regions defining CDR1, CDR2 and CDR3 respectively are identified in boxes. The unboxed sequences represent immunoglobulin framework. The length of shorter CDRs is adjusted for the alignment purpose by introducing dashes (-);

Figure 3 schematically shows amino acid sequences of CDR1, CDR2 and CDR3 for each immunoglobulin Heavy Chain Variable Region shown in Figure 2;

Figure 4 schematically shows the amino acid sequences defining a complete immunoglobulin Light Chain (Kappa) Variable Region of the antibodies assigned as IR3, IR4, IR4A, IR8, IR10, and IR24. The amino acid sequences are aligned relative to each other where the regions defining CDR1, CDR2 and CDR3 respectively are identified in boxes. The unboxed sequences represent immunoglobulin framework. The length of shorter CDRs is adjusted for the alignment purpose by introducing dashes (-);

Figure 5 schematically shows amino acid sequences of CDR1, CDR2 and CDR3 for each immunoglobulin Light Chain (Kappa) Variable Region shown in Figure 4;

Figure 6 shows the cloning site into pcDNA3.1-Zeo-(+) using 5 '-end Aflll and 3 '-end Apal restriction sites;

Figure 7 shows the results of the extracellular staining measurements with IGF-1R fluorescently labeled antibodies of R1610 cells expressing IGF-1R receptor, R1610 parental cells and 293T parental cells; Figure 8A shows the results of dissociation only measurements by surface plasmon resonance and corresponding calculations for IR4A antibody binding to human IGF-IR receptor;

Figure 8B shows the results of dissociation and associations measurements by surface plasmon resonance and corresponding calculations for IR4A antibody binding to human IGF-IR receptor;

Figure 9A shows the results of dissociation only measurements by surface plasmon resonance and corresponding calculations for IR8 antibody binding to human IGF-IR receptor;

Figure 9B shows the results of dissociation and associations measurements by surface plasmon resonance and corresponding calculations for IR8 antibody binding to human IGF-IR receptor;

Figure 10A shows the results of dissociation only measurements by surface plasmon resonance and corresponding calculations for IR10 antibody binding to human IGF-IR receptor;

Figure 10B shows the results of dissociation and associations measurements by surface plasmon resonance and corresponding calculations for IR10 antibody binding to human IGF-IR receptor;

Figure 11 A shows the results of dissociation only measurements by surface plasmon resonance and corresponding calculations for IR24 antibody binding to human IGF-IR receptor; and

Figure 1 IB shows the results of dissociation and associations measurements by surface plasmon resonance and corresponding calculations for IR24 antibody binding to human IGF-IR receptor.

DETAILED DESCRIPTION OF THE INVENTION The invention is based, in part, upon the discovery of antibodies that specifically bind to human IGF-IR (NCBI GL4557665) and block signaling induced by its cognate ligand IGF-1 (NCBI GL4529932). The antibodies could be used for a variety of diagnostic and therapeutic applications and as research tools. The antibodies were selected for their ability to bind to human IGF-IR with high affinity, specificity and selectivity. An additional property that the antibodies have been tested for is their ability to block physiological responses normally induced by IGF-1. The responses include blocking of IGF-1 R phosphorylation upon IGF-1 stimulation in a cell-based assay format, and antibody induced IGF-IR cell surface downregulation.

Since the antibodies described herein are engineered on the basis of human sequences, all of them could be administered to humans directly. Depending on a particular application, the described antibodies could be used as targeting moieties for various payloads such as

radionuclides, drugs, toxins and other effector molecules. Certain features and aspect of the application of the invention are described in more details below.

I - ANTIBODIES AGAINST HUMAN IGF-IR

In one aspect, the invention provides for an isolated antibody that specifically binds to human IGF-IR. The antibody is comprised of (1) an immunoglobulin light chain variable region comprised of three CDRs and (2) an immunoglobulin heavy chain variable region comprised of three other CDRs. The CDRs are embedded into the immunoglobulin framework generated by less variable FR domains. The CDRs of the immunoglobulin light and heavy chain brought together in immunoglobulin molecule define a unique binding site that specifically binds to a native conformation of IGF-IR receptor. The terms "binds specifically" or "specifically binds" are interchangeable and mean that binding affinities (EC50 values) of the antibodies described herein are below 50 nM (5* 10^~8 M).

It is understood that the antibodies can comprise both immunoglobulin heavy and light chain sequences of fragments thereof, such as Fab or Fab₂ fragments. It is understood that specific binding and functional properties can be displayed by a full-length intact immunoglobulin or antigen binding fragment thereof or biosynthetic antibody site.

It is understood that each of the antibody molecules can be an intact antibody, for example, a monoclonal antibody. Alternatively, the antigen binding could be displayed by an antigen binding fragment of an antibody or can be a biosynthetic antibody binding site. Antibody fragments include Fab, Fab₂ or Fv fragments. Techniques for making such antibody fragments are known to those skilled in the art. A number of biosynthetic antibody binding sites are known in the art and include single Fv or sFv molecules, for example as described in US Patent # 5,476,786. Other biosynthetic antibody binding sites include bi-specific or bi-functional antibodies that bind to at least two different target molecules. For example, a bi-specific antibody can bind to human IGF- 1R and to another antigen of interest. Methods for making bi-specific antibodies are known in art and include fusing hybridomas or linking Fab fragment together.

II - PRODUCTION OF IGF-1R ANTIBODIES

Antibodies described in this invention can be produced in different ways utilizing previously developed approaches. For example, DNA encoding variable regions of light and heavy chains can be synthesized chemically using commercially available services and sequence information provided in this invention. Alternatively, the DNA encoding variable regions of heavy and light chains can be amplified by Polymerase Chain Reaction (PCR) using the original clones of Fab fragments of the antibodies described herein, as templates. Synthetic or PCR-amplified DNA fragments can be genetically fused with appropriate nucleotide sequences to generate full-size antibodies or fragments thereof. Antibody expression constructs can be generated by including immunoglobulin constant region coding sequences, sequences providing expression control and other standard elements of expression systems. Generation of specific gene expression constructs is within ordinary skill in the art.

The DNA sequences encoding antibodies of interest can be genetically inserted into expression vectors that can be introduced into host cells using standard transfection of

transformation procedures known in the art. Examples of expression approaches include bacterial expression (E. coli) or mammalian expression (Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney (COS) cells, Human Embryo Kidney (HEK- 293) cells and myeloma cells that do not produce endogenous immunoglobulins. Transfected or transformed host cells can be propagated under conditions providing expression of genes of interest, such as immunoglobulin light and heavy chains and fragments thereof. The expressed proteins can be harvested using common techniques known in the art.

The particular conditions for production of antibodies or fragments thereof vary depending on the expression system utilized. For example, E. co/z^'-based expression system is particularly suitable for production of Fab, Fab₂ or sFv antibody fragments. The engineered antibody gene is cloned into a vector suitable for bacterial expression downstream from a commonly used bacterial promoters, e.g. T5 of Lac. Genetic fusion of a signal sequence providing targeting on the expressed protein into the periplasm may enable production and accumulation of soluble forms of antibody fragments into the periplasm of bacterial cells. Extraction of proteins of interest and, specifically, of antibody fragments from the periplasm of bacteria is a well-established array of standard methods known in the art.

If the antibodies are produced in mammalian expression systems, DNA coding sequences must be inserted into appropriate expression vectors containing adequate eukaryotic promoter, signal peptide for secretion from the cells and other genetic elements known in the arts.

Mammalian expression systems are particularly suitable for production of full-size

immunoglobulins. One of the approaches for antibody production is transient co-expression of heavy (variable + constant) and light (variable + constant) chains of immunoglobulin genetically introduced into two separate expression vectors. Another approach for antibody production is expression on both heavy and light chains from a single bi-cistronic vector. Alternatively, stable cell lines constitutively expressing both heavy and light chains can be generated using single vector approach or utilizing two-vector systems.

Alternative approaches for antibody production include expression in yeast (P. Pastoris or similar strains) or plant cells. Each expression system requires generation of host-specific genetic constructs and generation of expression constructs is very similar to described above bacterial and mammalian expression systems.

It is understood that regardless of the expression system utilized for production of antibodies of fragments thereof, the protein sequence of each antibody remains the same and directly corresponds to the sequence of this invention. It is also understood that DNA sequence may be altered, for example, by the process known as codon optimization that provides higher protein production, depending on host-specific codon usage.

It is understood that during the process of generation of expression constructs for antibodies and fragments thereof, various genetic modifications can be introduced. For example, epitope or purification tags or peptide and protein toxins can be genetically fused to the expression constructs encoding heavy chain, light chain or combination of both immunoglobulin chains.

Ill - MODIFICATIONS of IGF-1R ANTIBODIES It is understood that the antibodies disclosed herein can be modified to improve performance which largely depends on the intended use. For example, if used as therapeutic agent, the antibody can be genetically modified to reduce its immunogenicity in the intended recipient. Additionally, or as an alternative, the antibody can be genetically fused or coupled to another peptide or protein, such as epitope tag, purification tag, a growth factor, cytokine or natural or modified toxin. These modifications can be readily achieved by utilizing genetic manipulations known in the art.

IV - USE OF IGF-IR ANTIBODIES

The IGF-IR -specific antibodies described herein can be used as therapeutic agents, and diagnostic agents or as reagents for basic and applied research and development.

(1) Therapeutic Applications

Because the antibodies in the invention specifically bind to human IGF-IR receptor and block signaling mediated by its cognate ligand IGF-1, they can be utilized in a variety of therapeutic applications. The antibodies can be used to reduce or inhibit proliferation of tumor cells growth of which depends on IGF-IR / IGF-1 signaling. In this approach, tumor cells are exposed to a therapeutic concentration of the antibody to reduce or inhibit proliferation of the tumor cells. In certain embodiment, the antibodies inhibit tumor cell proliferation of at least 60%, 70%, 80%, 90% or 98%.

It is contemplated that the antibodies of the invention can be used for the treatment of a variety of disorders in which IGF-IR / IGF-1 axis is involved. This includes cancers originating from different types of tissues and organs such as breast cancer, lung cancer, head and neck cancer, esophageal cancer and some others.

It is contemplated that the antibodies of the invention can be utilized for the treatment of autoimmune diseases such as Multiple Sclerosis, Rheumatoid Arthritis, Inflammatory Bowl Disease and alike.

(2) Diagnostic Applications Whenever the antibodies are used for diagnostic purposes, either in vivo or in vitro, the antibodies are typically modified directly or indirectly with a detection moiety. The detection moiety is a functional addition to the antibody that can be detected either directly or indirectly or is capable of generating a detectable signal. For example, the detectable moiety can be a

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radionuclide ( Iodine, Phosphorus, Carbon or others), fluorescent or chemiluminescent compound, such as fluorescein, rhodamine or luciferine. Enzyme moieties include alkaline phosphatase, horse radish peroxidase, beta-galactozidase and others. Methods for conjugation of the detection moieties largely depend on the nature of the moiety and routinely can be reproduced by those experienced in the art.

The antibodies of the invention can be used in a broad range of immunological techniques know in the art. Examples of such techniques include sandwich immunoassays (ELISA), competitive immunoassays, cell surface staining procedures combined with FACS analysis, immunocyto- and immunohistochemical procedures. Protocols and method all of these procedures and assays are well-established and can be routinely carried out by those skilled in the art.

EXAMPLES

The following examples illustrate selection, identification and characterization of a number of fully human recombinant antibodies against human IGF-IR receptor.

Example 1 - Generation of Magnetic Proteo liposomes as antigen presenting platforms.

Isolation of antibodies that recognize native conformations of IGF-IR is absolutely critical for the development of such antibodies as therapeutics. In the human body and tissues IGF-IR is present in its native form, therefore only the antibodies that bind to the native receptor have practical utility. Preparation of antigen-presenting platforms that provide oriented and functional receptor in its native conformation is far from being trivial. Many methods of antibody generation, for example generation of antibodies against peptide epitopes or receptor fragments, usually are not successful for complex targets such as IGF-IR. Previously patented technology that relies on the generation of magnetic proteoliposomes (MPLs) (Sodroski, J.G. and T. Mirzabekov,

Proteoliposomes containing an integral membrane protein having one or more transmembrane domains; US Patent 6,761,902;) has been used for isolation of antibodies against IGF-IR. The main advantage of the core technology that relies on usage of MPLs is the ability to present highly purified and concentrated antigen (human IGF-IR in this invention) properly oriented and, most importantly, in its native conformation and functional state. Although the technology disclosed in US Patent 6,761,902 can be applied to a variety of complex membrane proteins, each target requires extensive optimization of protein extraction and MPL formation conditions.

The condition for extraction of human IGF-IR were tested and optimized prior to the initiation of the antibody selection procedure. An extensive matrix of combinations of various detergents, salts and buffer components was analyzed to identify conditions providing a balance between effective extraction of the IGF-IR and retaining of its function and native conformation. The IGF-IR functionality was tested by assaying binding of its ligand, IGF-1, to the IGF-IR immobilized on the surface of the MPLs. Stability of the MPL particles (as judged by IGF-1 binding) was also tested to ensure that the native conformation of the IGF-IR is retained for the duration of the antibody selection protocol.

Example 2 - Antibody libraries and selection of anti-IGF-lR antibodies.

Selection of human IGF-lR-specific antibodies was carried out from antibody libraries encoding a series of fully human Fab antibody fragments consisting of 10¹⁰ - 10¹¹ independent antibody clones. Randomization of all three CDRs, CDRl, CDR2 and CDR3 was carried out for the heavy chain of Fab fragments. Randomization of CDR3 was carried out for the light chain of Fab fragments whereas CDRl, CDR2 were kept invariant. A phage display library represents a collection of individual phage particles that express only a certain type of a genetic fusion of an individual Fab antibody fragment with a surface protein intrinsic for this particular type of phage. A fraction of the phage display library usually containing 10¹²-10¹³ phage particles is used as a primary source of the antibody variety.

A fraction of the phage display library (10¹²-10¹³ phage particles) was incubated with MPL preparations containing functional IGF-IR in its native conformation. The phage particles that did not bind to the IGF-lR-MPLs were removed by a series of subsequent washes under conditions providing retention of the native conformation of IGF-IR. The pool of phage particles that was bound to IGF-lR-MPLs was removed by acidic elution. The deconvo luted phage output (usually 10⁶-10⁸ phage particles) was harvested and further amplified by propagation in E. coli. The pool of the amplified phage is then used further for the second selection round as described for the first round above. A minimum of 2 and maximum of 4 selection rounds are carried out for a standard selection procedure.

Example 3 - Screening of anti-IGF-lR antibodies.

This example describes procedure for screening antibodies specific against native

conformations of IGF-1R receptor.

The screening procedure is based on the usage of live cells expressing human IGF- 1 R receptor on their surface. Generation of stable cell lines expressing IGF-1R is described in Example 6 below. R1610 cells expressing IGF-1R were used for screening.

Phage outputs from 3^rd or 4^th selection rounds (Example 2) represented pools of page particles that were used to infect E. coli to produce phagemid DNA. The phagemid DNA was digested with Nhel and BstEII to excise the entire Fab fragment and further introduced (ligated) into pQE3-Kan expression vector (Quiagen) digested with the same restriction enzymes, Nhel- BstEII. The resulting genetic construct was suitable for expression of Fab antibody fragment in bacteria under control of T5 promoter.

Pool of pQE3-Kan vector with inserted Fab fragments was used for transformation of E. coli. The individual colonies resulting from the transformation were picked up and propagated in 96- well plates. Expression of Fab fragments was induced by IPTG and the expressed protein was harvested using standard biochemical approaches know to those in the art. Each well of 96-well plate containing single Fab clone was harvested separately and used further for binding property testing. A minimum of 500 and maximum of 50,000 individual clones were usually analyzed in the screening procedure.

The cells expressing human IGF-1R were mixed with individual Fab preparations and allowed to interact for 30 min at 4°C. Unbound material was removed by pelleting the cells and by aspirating the supernatant. Binding the Fab antibody fragments to the cells was analyzed by secondary antibodies conjugated to phycoerythrin. The antibody binding was quantified by FACS analysis using 96-well plate compatible Guava flow cytometer. Clones that gave increase of the signal >5 fold over the background were scored as positive and kept for further analysis. The antibody clones that bound to IGF-1R were re -tested for their ability to interact with parental cells that did not express IGF-1R in order to identify specific IGF-1R binders. The test was performed as described above and the final candidates from the screening had the following properties: they bind to the IGF-lR-expressing cells but not the IGF-lR-negative parental cells.

Example 4 - Sequencing of anti-IGF-lR antibodies.

The candidate Fab antibody clones identified in Example 3 were subjected to a sequencing analysis to identify independent clones. Bacterial cultures were submitted to Beckman Genomics automated sequencing facility.

The experimental sequencing data were analyzed by appropriate software, specifically the nucleic acid sequences were aligned to identify identical clones and to determine the differences in the deduced protein sequences. For example, variable regions can be identified using IMGT ACQUEST webserver-based software

The sequencing analysis enabled detection of clones with identical sequences and only clones with unique and distinct DNA and protein sequences were kept for further evaluation.

In order to create a complete heavy chain and light chain immunoglobulin sequences, the variable region of the sequences shown below are combined with the corresponding constant region sequences. Signal peptides are not presented in the sequence; each sequence starts from the actual beginning of the antibody molecule according to Kabat numbering system.

(1) IR3 Variable Region, Heavy Chain (Seq ID NO. 02)

1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG

61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT AATTATAGTA TGAATTGGGT GCGTCAGGCG

121 CCGGGCAAAG GTCTGGAATG GGTTAGCGAT ATTGGTCCGC GGTTTAGCGT TACCAATTAT

181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC

241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGATCCA G

301 AGGCAGTTCG ACTACTGGGG CCAGGGAACC TTGGTCACCG TCTCGAGT (2) IR4 Variable Region, Heavy Chain (Seq ID NO. 04)

1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG

61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT AATTATTCTA TGAACTGGGT GCGTCAGGCG

121 CCGGGCAAAG GTCTGGAATG GGTTAGCGGG ATTTGGCCGG CTTTGGGCGT TACCTTTTAT

181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC

241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGCTCTGTT

301 TTCTTCTCTT ACAAATCTGG CTTCGACTAC TGGGGCCAGG GAACCTTGGT CACCGTCTCG

361 AG

(3) IR4A Variable Region, Heavy Chain (Seq ID NO. 06)

1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG

61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT AATTATTCTA TGAACTGGGT GCGTCAGGCG

121 CCGGGCAAAG GTCTGGAATG GGTTAGCGGG ATTTGGCCGG CTTTGGGCGT TACCTTTTAT

181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC

241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGCTCTGTT

301 TTCTTCTCTT ACAAATCTGG CTTCGACTAC TGGGGCCAGG GAACCTTGGT CACCGTCTCG

361 AGT

(4) IR8 Variable Region, Heavy Chain (Seq ID NO. 08)

1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG

61 AGTTGCGCGG CCAGTGGCTT TACCTTCACT AACTATGCTA TCCATTGGGT GCGTCAGGCG

121 CCGGGCAAAG GTCTGGAATG GGTTAGCTTT ATTTATCCGG CTGCGGGCTA TACCAACTAT

181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC

241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAGGTTCT

301 GTTTGGGACT ACGGCATGGA CTACTGGGGC CAGGGAACCT TGGTCACCGT CTCGAGT

(5) IR10 Variable Region, Heavy Chain (Seq ID NO. 10)

1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG

61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT AAATCTTCTA TGAGCTGGGT GCGTCAGGCG

121 CCGGGCAAAG GTCTGGAATG GGTTAGCAGT ATTGAGCCGA GGCTTGGCGT GACCGCGTAT

181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC

241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGAGGTATC

301 TCTTGGTGGG ACTACGCCAT GGACTACTGG GGCCAGGGAA CCTTGGTCAC CGTCTCGAGT (6) IR24 Variable Region, Heavy Chain (Seq ID NO. 12)

1 GAGGTGCAGC TGCTGGAGTC CGGTGGTGGT CTGGTACAGC CGGGTGGTTC TCTGCGTCTG

61 AGTTGCGCGG CCAGTGGCTT TACCTTCAGT TATTATGATA TCGCATGGGT GCGTCAGGCG

121 CCGGGCAAAG GTCTGGAATG GGTTAGCAAG ATTGGGCCGT ATGGTGGCGG GACCTACTAT

181 GCGGATAGCG TGAAAGGCCG TTTTACCATT TCTCGCGACA ACAGCAAGAA CACGCTGTAC

241 CTGCAGATGA ACTCACTGCG TGCCGAAGAT ACGGCCGTGT ATTACTGTGC GAGATCGTCG

301 CACCGCCGCG AGTACGCCTT TGACTACTGG GGCCAGGGAA CCTTGGTCAC CGTCTCGAGT

(7) IR3 Variable Region, Light Chain, Kappa (Seq ID NO. 32)

1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT

61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA

121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA

181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA

241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAAGGAAGT GGTATTCTAT CACGTTTGGC

301 CAAGGGACCA AGGTGGAAAT CAAACGTA

(8) IR4 Variable Region, Light Chain, Kappa (Seq ID NO. 34)

1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT

61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA

121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA

181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA

241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGATTATT ATTCTGATCC TGTCACGTTC

301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A

(9) IR4A Variable Region, Light Chain, Kappa (Seq ID NO. 36)

1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT

61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA

121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA

181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA

241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGCTTCTT CTTCTGATCC TATCACGTTC

301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A (10) IR8 Variable Region, Light Chain, Kappa (Seq ID NO. 38)

1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT

61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA

121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA

181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA

241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAAGGTGCTA TTCTTAGTCC TGTCACGTTC

301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A

(11) IR10 Variable Region, Light Chain, Kappa (Seq ID NO. 40)

1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT

61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA

121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA

181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA

241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATATTATT ATTCTCCTAT CACGTTCGGC

301 CAAGGGACCA AGGTGGAAAT CAAACGTA

(12) IR24 Variable Region, Light Chain, Kappa (Seq ID NO. 42)

1 GAAATTGTGC TGACCCAGTC TCCGGGCACG TTATCTCTGA GCCCTGGTGA GCGCGCCACT

61 CTGTCATGCC GGGCTTCTCA AAGTGTTAGC AGTAGCTACC TGGCGTGGTA TCAGCAAAAA

121 CCGGGCCAGG CCCCGCGTCT GCTGATTTAC GGTGCATCCA GCCGTGCCAC CGGCATTCCA

181 GATCGTTTTT CCGGTAGTGG TTCTGGGACG GACTTCACTC TGACAATCTC ACGCCTGGAA

241 CCGGAGGATT TTGCGGTGTA TTACTGCCAG CAATCTTATT CTTATTCTCC TTTCACGTTC

301 GGCCAAGGGA CCAAGGTGGA AATCAAACGT A

(13) Reference Human IgGl Heavy Chain Constant Region, nucleotide sequence

(Seq ID NO. 51)

0001 GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG

0141 CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC

0211 CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACC

0281 GTGCCCTCCA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG

0351 TGGACAAGAA AGTTGAGCCC AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTGAACT

0421 CCTGGGGGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT

0491 GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG

0561 GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG

0631 CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC 0701 CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC 0771 TGCCCCCATC CCGGGAGGAG ATGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC 0841 CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG

0911 CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA 0981 ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC 1051 TCCGGGTAAA TGA

(14) Reference Human IgGl Heavy Chain Constant Region, amino acid sequence

(Seq ID NO. 52)

001 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT 071 VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP 141 EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA 211 LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

281 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

(15) Reference Human Light Chain Kappa Constant Region, nucleotide sequence

(Seq ID NO. 53)

001 CCGGTCACCA TGGAAATCAA ACGTACGGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC 141 AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA 211 GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC

281 AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT 351 GCGAAGTCAC CCATCAGGGC CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG

(16) Reference Human Light Chain Kappa Constant Region, amino acid sequence

(Seq ID NO. 54)

001 PVTMEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD 071 STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC*

(17) Heavy Chain of the antibody IR3, full amino acid sequence (Seq ID NO. 55)

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYSMNWVRQA PGKGLEWVSD IGPRFSVTNY ADSVKGRFTI

71 SRDNSKNTLY LQMNSLRAED TAVYYCARSM RQFDYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA

141 LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSWTVPSS SLGTQTYICN VNHKPSNTKV

211 DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC WVDVSHEDP EVKFNWYVDG

281 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL

351 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN

421 VFSCSVMHEA LHNHYTQKSL SLSPGK (18) Heavy Chain of the antibody IR4, full amino acid sequence (Seq ID NO. 56)

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYSMNWVRQA PGKGLEWVSG IWPALGVTFY ADSVKGRFTI

71 SRDNSKNTLY LQMNSLRAED TAVYYCASSV FFSYKSGFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS

141 GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP

211 SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMI SRT PEVTCVWDV SHEDPEVKFN

281 WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

351 QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR

421 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

(19) Heavy Chain of the antibody IR4A, full amino acid sequence (Seq ID NO. 57)

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS NYSMNWVRQA PGKGLEWVSG IWPALGVTFY ADSVKGRFTI

71 SRDNSKNTLY LQMNSLRAED TAVYYCASSV FFSYKSGFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS

281 WYVDGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP

421 WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

(20) Heavy Chain of the antibody IR8, full amino acid sequence (Seq ID NO. 58)

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFT GSPKNWVRQA PGKGLEWVSF IYPAAGYTNY ADSVKGRFTI

71 SRDNSKNTLY LQMNSLRAED TAVYYCARGS VWDYGMDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG

141 TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN

211 TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMI SRTPE VTCVWDVSH EDPEVKFNWY

281 VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL ΡΑΡΙΕΚΤΙΞΚ AKGQPREPQV

351 YTLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ

421 QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

(21) Heavy Chain of the antibody IR10, full amino acid sequence (Seq ID NO. 59)

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS KSSMSWVRQA PGKGLEWVSS IEPRLGVTAY ADSVKGRFTI

71 SRDNSKNTLY LQMNSLRAED TAVYYCARGI SWWDYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG

141 GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS

211 NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW

281 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW

421 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (22) Heavy Chain of the antibody IR24, full amino acid sequence (Seq ID NO. 60)

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS YYDIAWVRQA PGKGLEWVSK IGPYGGGTYY ADSVKGRFTI 71 SRDNSKNTLY LQMNSLRAED TAVYYCARSS HRREYAFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG 141 GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS 211 NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW 281 YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 351 VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 421 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

(23) Light Chain Kappa of the antibody IR3, full amino acid sequence (Seq ID NO. 61)

1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT 71 DFTLTISRLE PEDFAVYYCQ QRKWYSITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF 141 YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF 211 NRGEC

(24) Light Chain Kappa of the antibody IR4, full amino acid sequence (Seq ID NO. 62)

1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT 71 DFTLTISRLE PEDFAVYYCQ QDYYSDPVTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASWCLLNN 141 FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS 211 FNRGEC

(25) Light Chain Kappa of the antibody IR4A, full amino acid sequence (Seq ID NO. 63)

1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT

71 DFTLTISRLE PEDFAVYYCQ QASSSDPITF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASWCLLNN

141 FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS 211 FNRGEC

(26) Light Chain Kappa of the antibody IR8, full amino acid sequence (Seq ID NO. 64)

1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT 71 DFTLTISRLE PEDFAVYYCQ QGAILSPVTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASWCLLNN 141 FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS 211 FNRGEC

(27) Light Chain Kappa of the antibody IR10, full amino acid sequence (Seq ID NO. 65)

1 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT

71 DFTLTISRLE PEDFAVYYCQ QYYYSPITFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF

141 YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF 211 NRGEC (28) Light Chain Kappa of the antibody IR24, full amino acid sequence (Seq ID NO. 66)

1 E IVLTQS PGT LSLS PGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLI Y GASSRATGI P DRFSGSGSGT

71 DFTLT I SRLE PEDFAVYYCQ QSYSYSPFTF GQGTKVEI KR TVAAPSVFI F PPSDEQLKSG TASWCLLNN

1 41 FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLS ST LTLSKADYEK HKVYACEVTH QGLSS PVTKS

2 11 FNRGEC

The sequences of Fab antibody fragments defining Variable Regions of immunoglobulin heavy chain selected from the phage display libraries as described in Example 2, screened as set forth in Example 3 and sequences of which identified in the Example 4 are shown in Figure 2. The sequences are aligned to each other due to homology of the antibody framework where CDRl, CDR2 and CDR3 are identified by boxes. Figure 3 shown an alignment of each CDR separately where dashes (-) are inserted to shorter CDRs for the alignment purpose.

The sequences of Fab antibody fragments defining Variable Regions of immunoglobulin light chain (kappa) selected from the phage display libraries as described in Example 2, screened as set forth in Example 3 and sequences of which identified in the Example 4 are shown in Figure 4. The sequences are aligned to each other due to homology of the antibody framework where CDRl, CDR2 and CDR3 are identified by boxes. Figure 5 shown an alignment of each CDR separately where dashes (-) are inserted to shorter CDRs for the alignment purpose.

Table 1 provides a correspondence between sequences discussed in these Examples with Sequence Listing (Seq. ID NO).

TABLE 1

Seq ID

Protein or Nucleic acid Description

NO

01 Heavy Chain Variable region of IR3 - Protein

02 Heavy Chain Variable region of IR3 - Nucleic acid

03 Heavy Chain Variable region of IR4 - Protein

04 Heavy Chain Variable region of IR4 - Nucleic acid

05 Heavy Chain Variable region of IR4A - Protein Seq ID

Protein or Nucleic acid Description

NO

06 Heavy Chain Variable region of IR4A - Nucleic acid

07 Heavy Chain Variable region of IR8 - Protein

08 Heavy Chain Variable region of IR8 - Nucleic acid

09 Heavy Chain Variable region of IR10 - Protein

10 Heavy Chain Variable region of IR10 - Nucleic acid

11 Heavy Chain Variable region of IR24 - Protein

12 Heavy Chain Variable region of IR24 - Nucleic acid

13 Heavy Chain CDR1 of IR3 - Protein

14 Heavy Chain CDR2 of IR3 - Protein

15 Heavy Chain CDR3 of IR3 - Protein

16 Heavy Chain CDR1 of IR4 - Protein

17 Heavy Chain CDR2 of IR4 - Protein

18 Heavy Chain CDR3 of IR4 - Protein

19 Heavy Chain CDR1 of IR4A - Protein

20 Heavy Chain CDR2 of IR4A - Protein

21 Heavy Chain CDR3 of IR4A - Protein

22 Heavy Chain CDR1 of IR8 - Protein

23 Heavy Chain CDR2 of IR8 - Protein

24 Heavy Chain CDR3 of IR8 - Protein

25 Heavy Chain CDR1 of IR10 - Protein

26 Heavy Chain CDR2 of IR10 - Protein

27 Heavy Chain CDR3 of IR10 - Protein

28 Heavy Chain CDR1 of IR24 - Protein

29 Heavy Chain CDR2 of IR24 - Protein

30 Heavy Chain CDR3 of IR24 - Protein

31 Light Chain (Kappa) Variable region of IR3 - Protein

32 Light Chain (Kappa) Variable region of IR3 - Nucleic acid

33 Light Chain (Kappa) Variable region of IR4 - Protein

34 Light Chain (Kappa) Variable region of IR4 - Nucleic acid Seq ID

Protein or Nucleic acid Description

NO

35 Light Chain (Kappa) Variable region of IR4A - Protein

36 Light Chain (Kappa) Variable region of IR4A - Nucleic acid

37 Light Chain (Kappa) Variable region of IR8 - Protein

38 Light Chain (Kappa) Variable region of IR8 - Nucleic acid

39 Light Chain (Kappa) Variable region of IR10 - Protein

40 Light Chain (Kappa) Variable region of IR10 - Nucleic acid

41 Light Chain (Kappa) Variable region of IR24 - Protein

42 Light Chain (Kappa) Variable region of IR24 - Nucleic acid

43 Light Chain (Kappa) CDR1 of IR3; IR4; IR4A; IR8; IR10; IR24 - Protein

44 Light Chain (Kappa) CDR2 of IR3; IR4; IR4A; IR8; IR10; IR24 - Protein

45 Light Chain (Kappa) CDR3 of IR3 - Protein

46 Light Chain (Kappa) CDR3 of IR4 - Protein

47 Light Chain (Kappa) CDR3 of IR4A - Protein

48 Light Chain (Kappa) CDR3 of IR8 - Protein

49 Light Chain (Kappa) CDR3 of IR10 - Protein

50 Light Chain (Kappa) CDR3 of IR24 - Protein

51 Reference Human IgGl Heavy Chain Constant Region, nucleotide sequence

52 Reference Human IgGl Heavy Chain Constant Region, amino acid sequence

53 Reference Human Light Chain Kappa Constant Region, nucleotide sequence

54 Reference Human Light Chain Kappa Constant Region, amino acid sequence

55 Heavy Chain of the antibody IR3, full amino acid sequence

56 Heavy Chain of the antibody IR4, full amino acid sequence

57 Heavy Chain of the antibody IR4A, full amino acid sequence

58 Heavy Chain of the antibody IR8, full amino acid sequence

59 Heavy Chain of the antibody IR10, full amino acid sequence

60 Heavy Chain of the antibody IR24, full amino acid sequence

61 Light Chain Kappa of the antibody IR3, full amino acid sequence

62 Light Chain Kappa of the antibody IR4, full amino acid sequence Seq ID

Protein or Nucleic acid Description

NO

63 Light Chain Kappa of the antibody IR4A, full amino acid sequence

64 Light Chain Kappa of the antibody IR8, full amino acid sequence

65 Light Chain Kappa of the antibody IR10, full amino acid sequence

66 Light Chain Kappa of the antibody IR24, full amino acid sequence

67 The cloning site into pcDNA3.1-Zeo-(+)

68 IA-001 Direct primer

69 IA-002 Reverse primer

70 AC-001 Direct 'structural' primer for assembly of the epitope tag

71 AC-002 Reverse 'structural' primer for assembly of the epitope tag

72 AC-003 Direct PCR primer for assembly of the epitope tag

73 AC-004 Reverse PCR primer for assembly of the epitope tag

74 Signal peptide for immunoglobulin secretion

75 A-370 Direct PCR primer for Variable Region of Heavy Chain IgGl

76 A-371 Reverse PCR primer for Variable Region of Heavy Chain IgGl

77 A-340 Direct PCR primer for Variable Region of Light Chain Kappa

78 A-341 Reverse PCR primer for Variable Region of Light Chain Kappa

Example 5 - Production of recombinant human IGF-IR. This Example describes design and generation of expression constructs for inducible expression of human IGF-IR receptor. In particular, this example describes epitope and purification tags genetically fused to the nucleic acid sequences encoding IGF-IR and its orthologs.

Human IGF-IR receptor (NCBI Reference Sequence: NM_000875.3) was amplified by PCR using Clone ID HsC QQ38509 (DNASU Plasmid Repository) as a template. To the coding sequence of the IGF-IR, Nhel restriction site for cloning purpose and Kozak sequence was genetically fused upstream of the starting native ATG codon using IA-001 Direct primer. The endogenous native Stop codon of the IGF-IR was removed using IA-002 Reverse primer to provide in- frame genetic fusion with Strep-tag (http: y^v^'.iba-go.de/prottoois/prot streptag.html. a 8-amino acid peptide tag) and FLAG epitope tag for detection purpose and introduced Aflll restriction site for cloning purpose. The PCR product obtained from the reaction using IA-001 and IA-002 primers and human IGF-1R as a template, was digested Nhel-Aflll and cloned into pcDNA3.1 -FlagStr vector (Invitrogen) .

Cloning of pcDNA3.1 -FlagStr vector was performed by PCR assembly of the epitope tag: - Link-FLAG-SBP-* (SBP = streptavidin binding peptide), containing Apal site for cloning.

Combination of AC-001, AC-002 'structural' primers and AC-003 and AC-004 Direct and Reverse PCR primers are required for assembly of the tag. The cloning site into pcDNA3.1-Zeo- (+) using 5 '-end Aflll and 3 '-end Apal restriction sites is shown in Figure 6.

IA-001 Direct primer. Nhel restriction site and Kozak sequence.

5'- ACTGCTAGCCATGAAGTCTGGCTCCGGAGGA -3' (SEQ ID NO. 68)

IA-002 Reverse primer. Aflll restriction site added and the Stop codon removed.

5'- ACTCTTAAGGCAGGTCGAAGACTGGGGCA -3' (SEQ ID NO. 69)

AC-001 Direct 'structural' primer for assembly of the epitope tag

5 ' - AGCGGGTCCTCTGGAGGGGGAGACTATAAGGATGACGATGACAAGA

GTATGGATGAGAAAACGACAGGTTGGCGCGGCGGGCATGTCGTT - 3 '

(SEQ ID NO. 70)

AC-002 Reverse 'structural' primer for assembly of the epitope tag

5 ' - GTTCCCGCTGGCCCTGAGGGTGGTGCTCCAATCTAGCCCTGAGTTGTT

CCAGCTCACCGGCCAGTCCTTCAACGACATGCCCGCCGCGCC -3 ' (SEQ ID NO. 71) AC-003 Direct PCR primer for assembly of the epitope tag

5 ' - ATCCTTAAGGGCAGCGGGTCCTCTGGAGGGGGAG -3 ' (SEQ ID NO. 72) AC-004 Reverse PCR primer for assembly of the epitope tag

5 ' - TATGGGCCCTAAGGTTCCCGCTGGCCCTGAGGG - 3 ' (SEQ ID NO. 73)

Example 6 - Generation of stable cell lines expressing recombinant IGF-1R.

The expression constructs encoding human IGF-1R receptor described in Example 5 were used for generation of stable cell lines. Commercially available cell lines, R1610, Cf2Th and HEK-293 (ATCC) were used. The expression constructs were verified for the expression of the protein of interest in a transient transfection experiment and then the cells were propagated on a medium containing Zeocin to select for stable cell lines harboring the gene of interest. Expression of the IGF-1R was verified by Western blot and by FACS analysis to ensure that the expressed protein was translocated to the plasma membrane. For both techniques, commercially available antibodies were used. All steps of the cell line generation were carried out according to the manufacturer' s protocol.

Example 7 - Conversion of the Fab antibody fragments into immunoglobulins and their production. This Example provides description and approach for subcloning of Fab fragments into mammalian expression vectors for production of fully functional immunoglobulins. The protein production approach is also provided herein.

The candidate Fab antibody Heavy Chain variable region fragments described in

EXAMPLES 2, 3 and 4 were converted into full size immunoglobulins of IgGl framework. Variable Region of heavy chain was fused to the constant region of human IgGl isotype using expression vector pTT-5 (NRC Biotechnology Research Institute, National Research Council of Canada) modified by introduction of the constant region of human IgGl from pFUSE-CHIg-hGl expression vector (Invivogen) resulting in pTT-IgGl-HC. The signal peptide from

immunoglobulin kappa light chain variable region (Mus musculus.gblAAG3571.8.! IAF20770S 1) was introduced into the construct upstream of the antibody variable sequences reported in this invention. Immunoglobulin 20-amino acid signal peptide sequence where starting methionine is underlined

METDTILLWVLLLWVPGSTG (SEQ ID NO. 74)

The variable regions of the heavy chain were amplified by PCR to introduce cloning sites as follows: 5 '-end cloning restriction site is Sail, 3 '-end restriction site is Nhel. The set of two primers, A-370 Direct primer and A-371 Reverse primer, was used for the PCR amplification. The resulting PCR fragment was digested with Sail -Nhel restriction enzymes and then introduced into pTT-IgGl-HC vector digested with the same enzymes.

A-370 Direct PCR primer for amplification of Heavy Chain Variable Region. Sail restriction site is underlined

5 ' -TGTGTCGACCGGAGAAGTTCAACTGCTGGAGTCCGGTGGTGGTCTGGTACAGCCGGGT GGTTCTCTG CGTCTGAGTTGCG-3' (SEQ ID NO. 75)

A-371 Reverse PCR primer for amplification of Heavy Chain Variable Region. Nhel restriction site is underlined

5' -TTGTGCTAGCACTCGAGACGGTGACCAAGGTTCCCTGGCC-3' (SEQ ID NO. 76)

The candidate Fab antibody Light Chain variable region fragments described in EXAMPLES 2, 3 and 4 were converted into full size immunoglobulins of IgGl framework. Variable Region of light chain was fused to the constant region of human light chain kappa using expression vector pTT-5 (NRC Biotechnology Research Institute, National Research Council of Canada) modified by introduction of the constant region of human light chain fragmen from pFUSE2-CLIg-hk expression vector (Invivogen) resulting in pTT-LC-Kappa. The signal peptide from

immunoglobulin kappa light chain variable region (Mus musculus, gb|AAG35718. i |AF207705 I) was introduced into the construct upstream of the antibody variable sequences reported in this invention.

The variable regions of the light chain were amplified by PCR to introduce cloning sites as follows: 5 '-end cloning restriction site is Sail, 3 '-end restriction site is BsiWI. The set of two primers, A-370 Direct primer and A-371 Reverse primer, was used for the PCR amplification. The resulting PCR fragment was digested with Sall-BsiWI restriction enzymes and then introduced into pTT-LC-Kappa vector digested with the same enzymes.

A-340 Direct PCR primer for amplification of Light Chain Kappa Variable Region. Sail restriction site is underlined

5' -TGTGTCGACCGGGGAAATTGTGTTGACGCAGTCTCCG-3' (SEQ ID NO. 77)

A-341 Reverse PCR primer for amplification of Light Chain Kappa Variable Region. BsiWI restriction site is underlined

5 ' -ATGGTGCAGCCACCGTACGTTTGATTTCCACC-3 ' (SEQ ID NO. 78)

The antibodies in a format of human IgGl framework were produced using protocol for transfection of CHO-3E7 cells using LPEI MAX in shake flask cultures. CHO-3E7 cells provided by NRC Biotechnology Research Institute, National Research Council of Canada, were diluted to 0.8 xlO⁶ cells/ml 24 h before transfection. On the day of transfection cell density was adjusted to 2.0 to 2.2xl0⁶ cells/ml using complete FreeStyle™ CHO medium and cell viability was greater than 97%.

The working solution of PEI was prepared as follows. To the 450 ml Milli-Q water, 1500 mg PEI "MAX" was added and stirred until complete dissolution and a final concentration of 3 mg/ml. The initial pH of the solution was around 2.2 and then it was adjusted to pH 7.0 by NaOH. The final pH adjustments were made using HC1 and/or NaOH. The final volume of the solution was adjusted to 500 ml, filter-sterilize using a 0.22 μιη membrane and stored at -20°C.

Polyethylenimine "MAX" linear, MW 25 kDa (40 kDa nominal), 3 mg/ml stock solution in water, pH 7.0 (Polysciences Inc. cat# 24765-2) was mixed with purified and quantified plasmid DNA of interest. A260/A280 ratio (use 50 mM Tris-HCl pH 8.0 to dilute the plasmid DNA) was between 1.85 and 1.95. The cells were used in exponential growth phase, 2-2.2xl0⁶ cells/ml in CHO FreeStyle medium. DNA preparations (0.75mg/L) encoding for Heavy or Light chains of immunoglobulin were mixed with PEI in CHO FreeStyle medium at 1 :5 (w:w) ratio, the mixture was then incubated 8-10 min, add then added to culture. Volume of the transfection mixture was 1/10 of the final volume of the production culture.

Routinely, expression level for various immunoglobulins was from 20 to 100 mg/L. The immunoglobulin production was monitored by commercially available ELISA kit (Bethyl

Laboratories).

Purification of immunoglobulin preparations was carried out as follows. Tris-Gly cine-Native

Buffer, lOx, pH 8.5 (Boston Bioproducts; Cat# BP- 160) was added to the supernatant (1/10 volume/volume) containing IgG at a final concentration of 20-100 mg/L and gently mixed. The supernatant volume was evenly distributed into 50-ml centrifuge tubes, 50ml per tube. Protein A Plus Agarose (Pierce, Cat. # 22812), a wet pellet, was used at 1/100 ratio to the total volume of the supernatant material. The appropriate amount of Protein A Plus Agarose in was incubated with the IgG-containing supernatants at an orbital shaker for overnight. After the incubation, the Protein A Plus Agarose resin was harvested and placed into 15 -ml columns (Pierce) and then washed with 10 volumes of lx PBS, then with 10 volumes of 25mM Tris-HCl, 0.12M Glycine, 1.5M NaCl (pH 8.5), then with 10 volumes of TBS-Tween-20, then with 10 volumes of 20mM Sodium Citrate Buffer, 1M NaCl (pH 5.5) and the final wash with 10 volumes of 150mM Sodium Chloride without any buffer. The elution of bound immunoglobulins was carried out by Elution Buffer (0.1 M Glycine pH 3.0, 10% Sucrose, 150mM NaCl) that was added at ratio of 1 : 1 to the volume of Protein A Plus Agarose resin. The elution buffer was incubated with the Protein A Plus Agarose resin for 3 min, removed and then another portion of fresh Elution Buffer was added to the Protein A Plus Agarose resin. The eluted material was immediately neutralized by 0.5 M Sodium Citrate Buffer, pH 6.0, at the 1/10 volume ratio to the eluted volume. The concentration of the resulting immunoglobulin preparations was determined by measure optical density of the solution at 280nm in UV -transparent cuvettes where a mixture of 0.1 ml of 0.5 M Sodium Citrate, pH 6, with 1ml of the Elution Buffer was used as a Reference Buffer. To calculate the concentration of IgG, the following formula that provides IgG concentration in mg/ml, was used:

[IgG] = standard extinction coefficient for an IgG.

Example 8 - Determination of affinities (EC50 values) of IGF-1R antibodies

This example describes the method for determination of the affinities of the antibodies against human IGF-1R receptor and provides means of comparison of properties of different antibody clones.

Stable cell line over-expressing IGF-1R under inducible promoter (as described in Example 6) or parental cells were used in the experiments. The cells (10,000 per well) were incubated with subsequent 2.5-fold dilutions of IgG preparations in FACS buffer (lx PBS, 2% Fetal Calf Serum, 0.2% sodium azide), 20μ1 total volume per well of V-shaped 96-well plate, for 40 min at 4°C. The cells were washed twice with FACS buffer and then stained with anti-human-Fc antibody-PE conjugate (Jackson Immunoresearch, #709-116-098) diluted 1 :50 in FACS. ΙΟμΙ of the diluted secondary conjugated antibody solution were added per well. Finally, cells were washed twice and fixated in FIX (lx PBS, 0.4% para-formaldehyde) at 75μ1 per well. The stained and fixated cells were analyzed by FACS using GUAVA PCA-96 flow cytometer at 425V. Mean Fluorescence Intensities (MFI) that reflect intensity of the antibody staining, were measured for each

experimental point in triplicates.

The affinities of the antibodies described in this invention are summarized in Table 2.

TABLE 2

Example 9 - Specificity of the IGF-1R antibodies.

Stable cell line over-expressing IGF-1R (as described in Example 6) as well as parental cells (R1610 and HEK293). The cells (10,000 per well) were incubated with IgG preparations at a final concentration of 100 nM in FACS buffer (lx PBS, 2% Fetal Calf Serum, 0.2% sodium azide), 20μ1 total volume per well of V-shaped 96-well plate, for 40 min at 4°C. The cells were washed twice with FACS buffer and then stained with anti-human-Fc antibody-PE conjugate (Jackson Immunoresearch, #709-116-098) diluted 1 :50 in FACS. ΙΟμΙ of the diluted secondary conjugated antibody solution were added per well. Finally, cells were washed twice and fixated in FIX (lx PBS, 0.4% para-formaldehyde) at 75 μΐ per well. The stained and fixated cells were analyzed by FACS using GUAVA PCA-96 flow cytometer at 425V. Mean Fluorescence Intensities (MFI) that reflect intensity of the antibody staining, were measured for each experimental point.

All cell lines were generated as described in the Example 6 using construct design and composition as described in the Example 5. The cell lines used in these experiments are listed in Table 3. TABLE 3

1 R1610-human IGF-1R (Extracellular staining with the corresponding antibody and

secondary anti-human-Fc antibody-PE conjugate (Jackson Immunoresearch, #709- 116-098))

2 R1610 parental, (Extracellular staining with the corresponding antibody and secondary

anti-human-Fc antibody-PE conjugate (Jackson Immunoresearch, #709-116-098))

3 HEK293 parental, (Extracellular staining with the corresponding antibody and secondary

anti-human-Fc antibody-PE conjugate (Jackson Immunoresearch, #709-116-098))

All of the indicated cell lines were validated for the expression or non-expression of the IGF- 1R receptor by using commercially available fiuorescently labeled antibodies (as specified in Table 3) specific to the receptors. The results of extracellular staining measurements are shown in Figure 7 and summarized in Table 4.

TABLE 4

Example 10 -Receptor binding properties of the IGF-IR antibodies.

The binding properties of the antibodies of the present invention to the cognate receptor IGF- IR were assessed with the surface Plasmon resonance method utilizing ProteOn XPR36 instrument. 200 μΐ of IGF-IR receptor at a concentration of 50 μg/ml were used for attachment to a biotin-immobilized chip. The antibodies were tested at a concentration of 120 nM in a PBS buffer containing 0.01% Tween20 at a temperature of 25°C. Data analysis was performed utilizing a bivalent binding model appropriated for approximating antibody biding mode. Representative analysis of the results are presented in Figures 8 through 11.

Example 11 - Functional properties of the IGF-IR antibodies.

Functional properties of the antibody clones reported herein were studied to identify antibodies with antagonistic properties. MTT cytotoxicity assay was used as a test for growth inhibition caused by antibody induced IGF-IR blocking in MCF7 cells (ATCC) that endogenously express high amounts of functionally active IGF-IR receptor.

IGF-IR receptor is over-expressed on the surface of many different primary and cultured cancer cell lines. This receptor efficiently upregulates the proliferation efficiency of tumor cells. A neutralizing (blocking, inhibiting) IGF-IR antibody binds to IGF-IR receptor and inhibits cellular proliferation. Cancer cell cytotoxic effect of anti-IFG-lR antibodies is broadly used for cell based estimation of their efficacy.

MCF7 human breast cancer cells were plated in a rich media (DMEM, 10%FCS, IX pen/Strep) over night to promote an effective proliferation. After this anti-IFG-lR antibodies disclosed herein have been added to the media in different concentrations for 72 hours and their cytotoxic potential has been estimated by inhibition of proliferation. Cytotoxic responses on MCF7 human cancer cell line was followed with quantitative analysis of cell proliferation using the MTT assay, which is based on the measurements of the activities of intracellular dehydrogenases. The main goal of this study is to compare the functional properties of the new therapeutic antibody candidate compounds.

Analysis of direct inhibition of ligand mediated IGF-IR signaling was performed by Western blot analysis of extracts from MCF7 cells, untreated or stimulated with IGF- 1 , using Phospho-IGF- 1 Receptor β Antibody (Tyrl 135/1136)(19H7, Cell Signal). MCF7 cells responded to addition of IGF- 1 (1 OOng/ml for 5 minutes) assessed by IGF-IR phosphorylation in positive control versus negative control. IR8 was able to block IGF-IR phosphorylation upon stimulation with IGF- 1. Analysis of IGF-1R internalization was performed using flow cytometry analysis of surface IGF-1R presentation on MCF7 cells, untreated (Ctrl) or treated with antibodies disclosed herein at 50nM for 16h. After this the cells were stained with commercially available anti-IGF-lR antibodies (Cell Signaling). MCF7 cells responded to addition of the antibodies disclosed herein by decreasing the amount of surface IGF-1R.

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

claimed is:

An isolated antibody that binds human IGF-IR receptor comprising an immunoglobulin light chain of SEQ ID NO. 61;

and an immunoglobulin heavy chain of SEQ ID NO. 55; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-IR receptor comprising an immunoglobulin light chain of SEQ ID NO. 62;

and an immunoglobulin heavy chain of SEQ ID NO. 56; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-IR receptor comprising an immunoglobulin light chain of SEQ ID NO. 63;

and an immunoglobulin heavy chain of SEQ ID NO. 57; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-IR receptor comprising an immunoglobulin light chain of SEQ ID NO. 64;

and an immunoglobulin heavy chain of SEQ ID NO. 58; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-IR receptor comprising an immunoglobulin light chain of SEQ ID NO. 65;

and an immunoglobulin heavy chain of SEQ ID NO. 59; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-IR receptor comprising an immunoglobulin light chain of SEQ ID NO. 66;

and an immunoglobulin heavy chain of SEQ ID NO. 60; or an antigen binding fragment of the antibody.

7. The antibody of claim 1 , wherein the antibody is a monoclonal antibody.

8. The antibody of claim 2, wherein the antibody is a monoclonal antibody.

9. The antibody of claim 3, wherein the antibody is a monoclonal antibody

10. The antibody of claim 4, wherein the antibody is a monoclonal antibody.

11. The antibody of claim 5, wherein the antibody is a monoclonal antibody.

The antibody of claim 6, wherein the antibody is a monoclonal antibody

An isolated antibody that binds human IGF-IR receptor comprising

(i) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 45; and

(ii) an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 13, a CDRH2 comprising the sequence of SEQ ID NO. 14, and a CDRH3 comprising the sequence of SEQ ID NO. 15; or an antigen binding fragment of the antibody.

14. An isolated antibody that binds human IGF-IR receptor comprising

(i) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 46; and

(ii) an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 16, a CDRH2 comprising the sequence of SEQ ID NO. 17, and a CDRH3 comprising the sequence of SEQ ID NO. 18; or an antigen binding fragment of the antibody. An isolated antibody that binds human IGF-1R receptor comprising

(i) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 47; and

(ii) an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 19, a CDRH2 comprising the sequence of SEQ ID NO. 20, and a CDRH3 comprising the sequence of SEQ ID NO. 21; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-1R receptor comprising

(i) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 48; and

(ii) an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 22, a CDRH2 comprising the sequence of SEQ ID NO. 23, and a CDRH3 comprising the sequence of SEQ ID NO. 24; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-1R receptor comprising

(i) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 49; and

(ii) an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 25, a CDRH2 comprising the sequence of SEQ ID NO. 26, and a CDRH3 comprising the sequence of SEQ ID NO. 27; or an antigen binding fragment of the antibody.

An isolated antibody that binds human IGF-1R receptor comprising

(i) an immunoglobulin light chain variable region comprising a CDRL1 comprising the sequence of SEQ ID NO. 43, a CDRL2 comprising the sequence of SEQ ID NO. 44, and a CDRL3 comprising the sequence of SEQ ID NO. 50; and (ii) an immunoglobulin heavy chain variable region comprising a CDRHl comprising the sequence of SEQ ID NO. 28, a CDRH2 comprising the sequence of SEQ ID NO. 29, and a CDRH3 comprising the sequence of SEQ ID NO. 30; or an antigen binding fragment of the antibody.

19. The binding-protein antibody of claim 13 wherein the CDR sequences are interposed

between human or humanized framework sequences.

20. The binding-protein antibody of claim 14 wherein the CDR sequences are interposed

between human or humanized framework sequences.

21. The binding-protein antibody of claim 15 wherein the CDR sequences are interposed

between human or humanized framework sequences.

22. The binding-protein antibody of claim 16 wherein the CDR sequences are interposed

between human or humanized framework sequences.

23. The binding-protein antibody of claim 17 wherein the CDR sequences are interposed

between human or humanized framework sequences.

24. The binding-protein antibody of claim 18 wherein the CDR sequences are interposed

between human or humanized framework sequences.

25. An isolated antibody that binds human IGF-1R receptor comprising

an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 31, and

an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 01; or

an antigen binding fragment of the antibody.

26. An isolated antibody that binds human IGF-1R receptor comprising an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 33, and

an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 03; or

an antigen binding fragment of the antibody.

27. An isolated antibody that binds human IGF-1R receptor comprising

an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 35, and

an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 05; or

an antigen binding fragment of the antibody.

28. An isolated antibody that binds human IGF-1R receptor comprising

an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 37, and

an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 07; or

an antigen binding fragment of the antibody.

29. An isolated antibody that binds human IGF-1R receptor comprising

an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 39, and

an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 09; or

an antigen binding fragment of the antibody.

30. An isolated antibody that binds human IGF-1R receptor comprising

an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO. 41, and an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 11; or

an antigen binding fragment of the antibody.

31. The antibody of claim 25, wherein the antibody is a monoclonal antibody.

32. The antibody of claim 26, wherein the antibody is a monoclonal antibody.

33. The antibody of claim 27, wherein the antibody is a monoclonal antibody.

34. The antibody of claim 28, wherein the antibody is a monoclonal antibody.

35. The antibody of claim 29, wherein the antibody is a monoclonal antibody.

36. The antibody of claim 30, wherein the antibody is a monoclonal antibody.