KR20070001883A - Anti-igf-i receptor antibody - Google Patents

Abstract

Antibodies, humanized antibodies, resurfaced antibodies antibody fragments, derivatized antibodies, and conjugates of same with cytotoxic agents, which specifically bind to, and inhibit, insulin-like growth factor-I receptor, antagonize the effects of IGF-I, IGF-II and serum on the growth and survival of tumor cells, and which are substantially devoid of agonist activity. The antibodies and fragments thereof may be used, optionally in conjunction with other therapeutic agents, in the treatment of tumors that express elevated levels of IGF-I receptor, such as breast cancer, colon cancer, lung cancer, ovarian carcinoma, synovial sarcoma, prostate cancer and pancreatic cancer and the derivatized antibodies may be used in the diagnosis and imaging of tumors that express elevated levels of IGF-I receptor. ® KIPO & WIPO 2007

Description

ANTI-IGF-I RECEPTOR ANTIBODY

This application is part of CIP application on file No. 10 / 170,390, filed Jun. 14, 2002 and incorporated by reference herein in its entirety.

The present invention relates to antibodies that bind to human insulin-like growth factor-I receptors. More specifically, the present invention relates to anti-IGF-I receptor antibodies that inhibit the cellular function of the IGF-I receptor. Even more specifically, the present invention relates to anti-IGF-I receptor antibodies which oppose and rarely antagonize the effects of IGF-I, IGF-II and serum on the growth and survival of tumor cells. The invention also relates to fragments of the antibodies, humanized and resurfaced forms of the antibodies, conjugates of the antibodies, antibody derivatives, and the use of antibody derivatives for diagnostic, research, and therapeutic use. The present invention further relates to improved antibodies or fragments thereof made from the antibodies and fragments thereof described above. In another aspect, the invention relates to a polynucleotide encoding an antibody or fragments thereof and a vector constituting the polynucleotide.

Insulin-like growth factor-I receptor (IGF-I receptor) is a heterotetramer membrane transmembrane protein, two extracellular alpha chains in the form of β-α-α-β and beta chains across the two membranes. Has Ligand binding by the extracellular domain of the IGF-I receptor is IGF-I and IGF-II, which activates intracellular tyrosine kinases, leading to receptor phosphorylation and substrate phosphorylation. The IGF-I receptor is homologous to the insulin receptor, having 84% high sequence proximity in the tyrosine kinase domain of β chain and 48% low sequence proximity in the extracellular cysteine rich domain of α chain (Ulrich, A. et al. , 1986, EMBO , 5, 2503-2512; Fujita-Yamaguchi, Y. et al., 1986, J. Biol. Chem . , 261, 16727-16731; LeRoith, D. et al., 1995, Endocrine Reviews , 16 , 143-163). IGF-I receptors and their ligands (IGF-I and IGF-II) play important roles in many physiological processes, including growth and development during embryonic development, metabolism in adults, cell proliferation, and cell differentiation ( LeRoith, D., 2000, Endocrinology , 141, 1287-1288; LeRoith, D., 1997, New England J. Med . , 336, 633-640).

IGF-I and IGF-II are widely present in complexes with IGF-binding proteins and act as endocrine hormones in the blood and as internally produced lateral and self-secreting growth factors (Humbel, RE, 1990, Eur . J.). . Biochem, 190, 445-462;. ... Cohick, WS and Clemmons, DR, 1993, Annu Rev Physiol 55, 131-153).

IGF-I receptors are associated with promoting growth, modification, and survival of tumor cells (Baserga, R. et al., 1997, Biochem . Biophys . Acta , 1332, F105-F126; Blakesley, VA et al. , 1997, Journal of Endocrinology , 152, 339-344; Kaleko, M., Rutter, WJ, and Miller, AD 1990, Mol . Cell . Biol . , 10, 464-473). Thus, several types of tumors, including breast cancer, colon cancer, ovarian tumor, synovial sarcoma, and pancreatic cancer, are known to express IGF-I receptors above normal levels (Khandwala, HM et al., 2000, Endocrine Reviews , 21, 215-244; Werner, H. and Le Royth, D., 1996, Adv . Cancer Res . , 68,183-223; Happerfield, LC et al., 1997, J. Pathol . , 183, 412-417; Frier, S. et al., 1999, Gut , 44, 704-708; van Dam, PA et al., 1994, J. Clin . Pathol . , 47, 914-919; Xie, Y. et al., 1999, Cancer Res . 59, 3588-3591; Bergmann, U. et al., 1995, Cancer Res. , 55, 2007-2011). In vitro, IGF-I and IGF-II have been known as potent mitogens against various human tumor cell lines, including lung cancer, breast cancer, colon cancer, osteosarcoma and cervical cancer (Ankrapp, DP and Bevan, DR, 1993). , Cancer Res. , 53, 3399-3404; Cullen, KJ, 1990, Cancer Res . , 50, 48-53; Hermanto, U. et al., 2000, Cell Growth & Differentiation , 11, 655-664; Guo, YS et al., 1995, J. Am . Coll . Surg . , 181, 145-154; Kappel, CC et al., 1994, Cancer Res . , 54, 2803-2807; Steller, MA et al., 1996, Cancer Res . , 56, 1761-1765). These various tumors and tumor cell lines also express high levels of IGF-I or IGF-II that stimulate their growth in a self-secreting or lateral secretory manner (Quinn, KA et al., 1996, J. Biol . Chem . , 271, 11477-11483).

Epidemiological studies have shown that elevated plasma values of IGF-I (and lower levels of IGF-binding protein-3) correlate with increased risk for prostate cancer, colon cancer, lung cancer and breast cancer (Chan , JM et al., 1998, Science , 279, 563-566; Wolk, A. et al., 1998, J. Natl . Cancer Inst. , 90, 911-915; Ma, J. et al., 1999, J. Natl . Cancer Inst . , 91, 620-625; Yu, H. et al., 1999, J. Natl . Cancer Inst . , 91, 151-156; Hankinson, SE et al., 1998, Lancet , 351, 1393-1396). Measures to lower the IGF-I plasma value or inhibit the action of the IGF-I receptor have been proposed for cancer prevention (Wu, Y. et al., 2002, Cancer Res . 62, 1030-1035; Grimberg, A and Cohen P., 2000, J. Cell . Physiol . , 183, 1-9).

IGF-I receptors protect tumor cells from apoptosis caused by growth factor deficiency, anchorage independence or cytotoxic drug therapy (Navarro, M. and Baserga, R., 2001, Endocrinology , 142, 1073-1081;... Baserga , R. et al, 1997, Biochem Biophys Acta, 1332, F105-F126). Domains of the IGF-I receptors essential for mitosis, modification and anti-apoptotic activity were specified in mutation analysis.

For example, the tyrosine 1251 site of the IGF-I receptor has been recognized as essential for anti-apoptosis and modifying activity but not for mitotic activity (O'Connor, R. et al., 1997, Mol . Cell . Biol . , 17, 427-435; Miura, M. et al., 1995, J. Biol . Chem . , 270, 22639-22644). Intracellular signaling pathways of ligand-activated IGF-I receptors are involved in the phosphorylation of tyrosine sites of insulin receptor substrates (IRS-1 and IRS-2), and phosphatidylinositol-3-kinase (PI-3-kinase) To Phospholipid products of PI-3-kinase bound to the cell membrane activate serine / threonine kinase Akt, and their substrates include the pro-apoptotic protein BAD, which is phosphorylated in an inactive state (Datta, SR, Brunet, A. and Greenberg). , ME, 1999, Genes & Development , 13, 2905-2927; Kulik, G., Klippel, A. and Weber, MJ, 1997, Mol. Cell . Biol . 17, 1595-1606). The mitotic signaling pathway of the IGF-I receptor in MCF-7 human breast cancer cells requires PI-3-kinase and is independent of mitotic-activated protein kinase, while differentiated rat chromophilocytoma Survival signaling pathways in PC12 cells require both PI-3-kinase and mitotic-activated protein kinase pathways (Dufourny, B. et al., 1997, J. Biol . Chem . , 272, 31163-31171; Parrizas , M., Saltiel, AR and Le Roith, D., 1997, J. Biol . Chem . , 272, 154-161).

Downregulation of IGF-I receptors by antisense technology has shown that in vivo and in vitro tumors of various tumor cell lines, including melanoma, lung tumors, ovarian cancer, glioblastoma, neuroblastoma and rhabdomyosarcoma Has been shown to reduce tumorigenicity (Resnicoff, M. et al., 1994, Cancer Res . , 54, 4848-4850; Lee, C.-T. et al., 1996, Cancer Res . 56, 3038-3041; Muller, M. et al., 1998, Int . J. Cancer , 77, 567-571; Trojan, J. et al., 1993, Science , 259, 94-97; Liu, X. et al., 1998, Cancer Res . , 58, 5432-5438; Shapiro, DN et al., 1994, J. Clin . Invest . 94, 1235-1242). In addition, dominant negative mutants of the IGF-I receptor have been shown to reduce the in vivo tumorigenicity and in vitro growth of modified Rat-1 cells overexpressing the IGF-I receptor ( Prager, D. et al., 1994, Proc . Natl . Acad . Sci . USA , 91, 2181-2185).

Tumor cells expressing antisense for the IGF-I receptor mRNA experience huge apoptosis when injected into animals in the biodiffusion chamber. This makes IGF-I receptors a major therapeutic target based on the hypothesis that tumor cells are more sensitive to apoptosis by IGF-I receptor inhibition than normal cells (Resnicoff, M. et al., 1995, Cancer Res . , 55, 2463-2469; Baserga, R., 1995, Cancer Res . , 55, 249-252).

Another method for inhibiting the action of IGF-I receptors in tumor cells has been to use anti-IGF-I receptor antibodies that bind to and inhibit the extracellular domain of the IGF-I receptor. Various attempts have been made to express mouse monoclonal antibodies against the IGF-I receptor, of which two inhibitory antibodies -IR3 and 1H7- are available and their use has been documented in various IGF-I receptor studies. .

IR3 antibodies were expressed using partially purified placental products of the insulin receptor to immunize mice, wherein one antibody IR3 and the IGF-I receptor (somatomedin-C receptor) selected to bind the insulin receptor were preferred. Two antibodies IR2 and IR3 were produced that showed weak immunoprecipitation of the insulin receptor as well as immunoprecipitation (Kull, FC et al., 1983, J. Biol . Chem . , 258, 6561-6566).

1H7 antibody was expressed by immunizing mice with placental products of purified IGF-I receptors, where three inhibitory antibodies were produced in addition to inhibitory antibody 1H7 (Li, S.-L. et al., 1993, Biochem .. Biophys Res Commun, 196, 92-98;....... Xiong, L. et al, 1992, Proc Natl Acad Sci USA, 89, 5356-5360).

Another report reported that mouse monoclonal antibody groups specific for human IGF-I receptors were obtained by expressing high levels of IGF-I receptors and immunizing mice with infected 3T3 cells, which examined binding competition and infected IGF-Is. It was classified into seven groups according to whether to inhibit or promote binding to 3T3 cells (Soos, MA et al., 1992, J. Biol . Chem . , 267, 12955-12963).

Thus, although IR3 antibodies are the most commonly used inhibitory antibodies in in vitro studies of IGF-I receptors, they have the problem of showing antagonistic activity against infected 3T3 and CHO cells expressing human IGF-I receptors. . (Kato, H. et al, 1993, J. Biol Chem, 268, 2655-2661;...... Steele-Perkins, G. and Roth, RA, 1990, Biochem Biophys Res Commun, 171, 1244- 1251). Similarly, the most inhibitory antibodies 24-57 and 24-60 among the antibody groups developed by Soos et al. Also showed antagonistic action on infected 3T3 cells (Soos, MA et al., 1992, J. Biol . Chem ., 267, 12955-12963). Although IR3 antibodies have been reported to inhibit IGF-I (but not IGF-II) binding to receptors expressed in intact cells and after solubilization, both in IGF-I and IGF-II in vitro It has been shown to inhibit the ability to promote intracellular DNA synthesis (Steele-Perkins, G. and Roth, RA, 1990, Biochem . Biophys . Res . Commun ., 171, 1244-1251). The binding epitope of the IR3 antibody was inferred from the Shimeric insulin-IGF-I receptor construct at positions 223-274 of the IGF-I receptor (Gustafson, TA and Rutter, WJ, 1990, J. Biol . Chem . , 265, 18663-). 18667; Soos, MA et al., 1992, J. Biol . Chem . , 267, 12955-12963).

The MCF-7 human breast cancer cell line is widely used to describe the in vitro growth action of IGF-I and IGF-II as a cell line model (Dufourny, B. et al., 1997, J. Biol . Chem . , 272 , 31163-31171). In MCF-7 cells, IR3 antibodies incompletely protect against about 80% of the promoting effect of externally added IGF-I and IGF-II in serum-free conditions. In addition, IR3 antibodies show no significant inhibitory effect (less than 25%) on MCF-7 cell growth under 10% serum conditions (Cullen, KJ et al., 1990, Cancer Res . , 50, 48-53). The fact that IR3 antibody weakly inhibits serum-promoting growth of MCF-7 cells in vitro is due to in vivo studies that IR3 antibody treatment does not significantly inhibit growth by xenografting MCF-7 in nude mice. May be related (Arteaga, CL et al., 1989, J. Clin . Invest . , 84, 1418-1423).

Tumor cells, such as MCF-7 cells, in weak antagonism of IR3 and other reported antibodies and in a more physiological serum-promoting environment (instead of promotion in serum-free conditions by externally added IGF-I or IGF-II) Because of its inability to strongly inhibit growth, there is a need for new anti-IGF-I receptor antibodies that strongly inhibit serum-promoting growth of tumor cells but do not show strong antagonism.

We have discovered and improved new antibodies that specifically bind to human insulin-like growth factor-I receptor (IGF-IR) at the cell surface. Antibodies and fragments have the property of inhibiting the cellular function of the receptor without the ability to activate the receptor itself. Thus, while antibodies known to specifically bind to and inhibit IGF-IR activate receptors even in the absence of IGF-IR ligands, antibodies or fragments of the present invention antagonize IGF-IR but have little anti-inflammatory action. I never do that. Furthermore, the antibodies and antibody fragments of the present invention are capable of producing a serum of at least 80% (which corresponds to a higher inhibition than that obtained using known anti-IGF-IR antibodies) of human tumor cells such as MCF-7 cells. While present, suppress it.

The present invention proceeds from a murine anti-IGF-IR antibody (herein EM164) characterized by amino acid sequences of both light and heavy chains, the identification of CDRs, the specification of surface amino acids, and their means of expression in recombinant form.

The gene sequence is shown co-located with the sequence of EM164 in FIG. 15. The comparison reveals that somatic mutations are possible, one in CDR1 of the light chain of EM164 and one in CDR2 of the heavy chain.

Antibody EM164 light and heavy chains, and humanized forms of primary amino acid and DNA sequences are disclosed herein. However, the scope of the present invention is not limited only to antibodies and fragments comprising such sequences. Instead, all antibodies and fragments that specifically bind to insulin-like growth factor-I receptors and antagonize the biological activity of the receptors but rarely act as antivirals will fall within the scope of the present invention. Thus, antibodies and antibody fragments differ from antibody EM164 or its humanized derivatives in the amino acid sequences, CDRs, light chains and heavy chains of its backbone and fall within the scope of the present invention.

CDRs of antibody EM164 have been identified by modeling and their molecular structures have been predicted. Although CDRs are important for epitope recognition, they are not essential for the antibodies and fragments of the invention. Thus, for example, antibodies and fragments having improved properties provided by affinity maturation of the antibodies of the invention are provided.

Antibody mimics as well as various antibodies and antibody fragments can be readily prepared by mutations, deletions and / or insertions in variable and constant region sequences flanking a particular set of CDRs. Thus, for example, different types of antibodies (Ab) can be generated for a given set of CDRs by substitution of various heavy chains, thereby for example IgG1-4, IgM, IgA1-2, IgD, IgE antibody classes And isotypes can be prepared. Similarly, artificial antibodies can be produced by embedding a given set of CDRs entirely within the synthetic structure within the scope of the present invention. The term "variable" is used herein to describe the variability of a particular moiety that differs from one antibody to another and used in the binding and specificity of a particular antibody to an antigen, respectively. However, variability is generally not only given through the variable portion of an antibody.

Variability is generally concentrated in three parts called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable regions. A highly conserved portion of the variable portion is referred to as the structural portion FR. The heavy and light chain variable regions each comprise four structures, which usually have a beta-fold structure, which are linked to three CDRs to form overhanging loop connections and sometimes form part of the beta-wind structure. . CDRs in each chain are placed in close proximity by the FR region and, together with CDRs from other chains, contribute to the antigen binding formation of the antibody (EA Kabat et al. Sequences of Proteins of Immunological Interest , fifth edition, 1991, NIH). The constant region does not directly participate in the binding of the antigen but shows various effector functions such as the action of the antibody on antibody-dependent cytotoxicity.

Humanized antibodies, or antibodies adopted by other mammals to counter transplant rejection, can be produced using various techniques, such as resurfacing and CDR grafting. Resurfacing techniques incorporate molecular modeling, statistical analysis, and mutations to tailor non-CDR surfaces of various sites to resemble known antibody surface regions of the target host. Strategies and methods for resurfacing antibodies and other methods for reducing the immunity of antibodies in other hosts are disclosed in US Pat. No. 5,639,641, which is incorporated herein by reference. In CDR grafting techniques, murine heavy and light chain CDRs are grafted into fully human structural sequences.

The invention also includes functional equivalents of the antibodies described herein. Functional equivalents have binding properties that are comparable to those of antibodies, and include, for example, fragments as well as antibodies that are chimeric, humanized and single chain. Methods of producing such functional equivalents are described in PCT Publication No. 93/21319, European Patent Application No. 239,400; PCT Application Publication No. 89/09622; European Patent Application No. 338,745; And European Patent Application EP # 332,424, each of which is incorporated by reference herein.

Functional equivalents include polypeptides having an amino acid sequence that is substantially identical to the amino acid sequence of the variable or hypervariable portion of the antibodies of the present invention. "Almost identically" used in an amino acid sequence is at least about 90% different from other amino acid sequences. , More preferably, a sequence having at least about 95% homology, FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).

Shimeric antibodies preferably have a constant region derived substantially or exclusively from the constant region of a human antibody and a variable region derived substantially or solely from the sequence of the variable region of a mammalian antibody other than human. Humanized antibodies are prepared, for example, by substituting the complementarity determining regions of mouse antibodies into human structures, and are shown in PCT Publication No. WO 92/22653. Humanized antibodies preferably have complementarity determining regions (CDRs) derived substantially or exclusively from relevant human antibody sites and constant and variable regions other than CDRs derived from substantial or solely derived from non-human mammals.

Functional equivalents also include single chain antibody fragments, also known as single chain antibodies (scFv). These fragments comprise at least one antibody variable heavy chain amino acid sequence (V _H ) fragment linked to at least one antibody variable light chain sequence (V _L ) by or without one or more intermediate linkers. Such linkers are short, selected to confirm that an appropriate three-dimensional fold occurs when the (V _L ) and (V _H ) sites are linked to maintain the target molecular binding specificity of the whole antibody from which the single chain antibody fragment is derived. Flexible peptides. In general, the carboxy terminus of the (V _L) or (V _H) sequence may be covalently linked with the amino acid terminus of a complementary (V _L) and (V _H) sequence by a peptide linker. Single chain antibody fragments can be generated by molecular cloning, antibody phage display libraries, or similar techniques. These proteins can be produced in prokaryotic cells, including eukaryotic cells or bacteria.

Single chain fragments comprise an amino acid sequence having at least one variable or complementarity determining regions (CDRs) among all antibodies described herein, but lacking some or all of the constant regions of the antibodies. These constant regions are not necessary for antigen binding but constitute a major part of the structure of the entire antibody. Single chain antibody fragments can thus overcome some of the problems associated with the use of antibodies comprising some or all of the constant regions. For example, single-chain antibody fragments tend not to perform undesirable interactions between biomolecules and heavy chain constant regions, or other undesirable biological activities. Single chain antibody fragments are also much smaller than the previous antibody, and thus have greater capillary permeability than the previous antibody, allowing the single chain antibody fragment to be located and bound more effectively at the target antigen binding site. Antibody fragments can also be produced on a relatively large scale, promoting their production in prokaryotic cells. In addition, relatively small single-chain antibody fragments appear to produce less immune response in the receptor than all antibodies.

Functional equivalents also include antibody fragments with binding properties that are the same or contrasting to that of the previous antibody. Such fragments may comprise either or both Fab fragments or F (ab ') ₂ fragments. Although fragments containing fewer than all of the complementarity determining regions, such as 3, 4, or 5 CDRs, are also functional, preferably antibody fragments include all six complementarity determining regions of all antibodies. In addition, functional equivalents may be incorporated or incorporated into any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and subclasses thereof.

Information about the amino acid and nucleic acid sequences described herein for the anti-IGF-I receptor antibody EM164 and its humanized variants can be used to develop other antibodies that bind to the human IGF-I receptor and inhibit its cellular function. have. Various studies have examined the effect of describing one or more amino acid changes based on information on properties such as binding and expression of primary antibody sequences at various positions within the antibody sequence (Yang, WP et al., 1995). , J. Mol . Biol . , 254, 392-403; Rader, C. et al., 1998, Proc . Natl . Acad . Sci . USA , 95, 8910-8915; Vaughan, TJ et al., 1998, Nature Biotechnology , 16, 535-539).

The primary antibody variants in these studies used methods such as oligonucleotide-mediated relocation mutations, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutant strains of E. coli . By modifying the heavy and light chain genes in CDR1, CDR2, CDR3, or structural regions (Vaughan, TJ et al., 1998, Nature Biotechnology , 16, 535-539; Adey, NB et al., 1996, Chapter 16, pp. 277-291, in " Phage Display of Peptides and Proteins " , Eds. Kay, BK et al., Academic Press) .Methods of modifying the sequence of primary antibodies resulted in improving the affinity of secondary antibodies (Gram, H. et al., 1992, Proc. ... Natl Acad Sci USA, 89, 3576-3580;.. Boder, ET et al, 2000, Proc Natl Acad Sci USA, 97, 10701-10705;.... Davies, J. and Riechmann, L., 1996, Immunotechnolgy , 2, 169-179; Thompson, J. et al., 1996, J. Mol . Biol . , 256, 77-88; Short, MK et al., 2002, J. Biol . Chem . , 277 , 16365-16370; Furukawa, K. et al., 2001, J. Biol . Chem . , 276, 27622-27628).

By strategies designed to modify amino acid residues of similar one or more antibodies, the antibody sequences described herein can be used to develop anti-IGF-I receptor antibodies with improved function.

The conjugates of the present invention are linked to cytotoxic materials to constitute antibodies, fragments, and their analogs as disclosed herein. Preferred cytotoxic substances are maytansinoids, taxanes and analogs of CC-1065. The conjugates can be prepared by in vitro methods. A linker is used to link the cytotoxic substance to the antibody. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid-labile groups, photo-labile groups, peptidase-labile groups and esterase-labile groups. Preferred linking groups are disulfide groups and thioether groups. For example, the conjugates can be prepared using disulfide exchange or by forming thioether bonds between the antibody and the cytotoxic substance.

Maytansinoids and their analogs are among the preferred cytotoxic substances. Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Suitable maytansinoids are described in US Pat. No. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; And 5,846,545.

Taxanes are also preferred cytotoxic substances. Taxanes suitable for use in the present invention are disclosed in US Pat. Nos. 6,372,738 and 6,340,701. CC-1065 and the like are also preferred cytotoxins for use in the present invention. CC-1065 and its analogues are described in US Pat. No. 6,372,738; 6,340,701; 5,846,545 and 5,585,499.

A suitable material for the preparation of cytotoxic conjugates is CC-1065, a potent anti-tumor antibiotic isolated from the culture of Streptomyces xerensis. CC-1065 is about 1000 times more potent in vitro than commonly used anticancer agents such as doxorubicin, mesotrexate and vincristine (BK Bhuyan et al., Cancer Res ., 42 , 3532-3537 (1982)).

Cytotoxins such as mesotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and calicheamycin are also suitable for preparing the conjugates of the present invention, the toxin molecules They can also be linked to antibody molecules through intermediate carrier materials such as serum albumin.

For diagnostic purposes, antibodies of the invention will typically be labeled with a detectable substance. The detectable substance can be any substance that can produce a detectable signal either directly or indirectly. For example, the detectable substance may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹³¹ I; Fluorescent or chemiluminescent compounds such as fluorescein isothiocyanate, rhodamine, or luciferin; Or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Hunter, et al., Nature 144: 945 (1962); David, et al., Biochemistry 13: 1014 (1974); Pain, et al., J. Immunol. Meth. 40: 219 (1981); And Nygren, J. Histochem. and Cytochem. Any technique known in the art, including those described by 30: 407 (1982), can be employed to conjugate the antibody to a detectable material.

Antibodies of the invention can be applied in known assay methods such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987). )).

Antibodies of the invention are also useful for in vivo imaging, wherein an antibody labeled with a detectable substance, such as a radio-impermeable substance or radioisotope, is administered to a host, preferably in the blood stream, and the labeled antibody is host It is observed to be present and located in. Such imaging techniques are useful for determining and treating the progression of malignant tumors. The antibody can be labeled with any detectable substance in the host, such as nuclear magnetic resonance, radiation, or other means of detection known in the art.

Antibodies of the invention are also useful as affinity purification materials. In this process the antibodies are immobilized using well known techniques on a suitable support means such as Sephadex resin or filter paper.

Antibodies of the invention are also useful as reagents for biological investigations based on the inhibition of the function of IGF-I receptors in cells.

For therapeutic use, the antibodies or conjugates of the invention are administered to a host in a pharmaceutically acceptable dosage form. It may be administered intravenously in the form of a pill or via continuous infusion, intramuscular, subcutaneous, inter-articular, intrasynovial, interdermal, oral, topical, or inhalational route over time. Antibodies can also be administered by intertumor, flank, wound, or wound flank routes to exert local therapeutic effects as well as the whole body. Suitable pharmaceutically acceptable carriers, diluents, and receptors are well known and can be determined as clinical stage clearance by those of ordinary skill in the art. Examples of suitable carriers, diluents and / or receptors include: (1) Dulbecco's phosphate buffer containing about 1 mg / ml to 25 mg / ml human serum albumin, pH about 7.4,, (2) 0.9% Salts (0.9% w / v NaCl), and (3) 5% (w / v) dextrose. The methods of the invention can be carried out in vitro, in vivo, or ex vivo.

In other therapeutic techniques, the antibodies, antibody fragments or conjugates of the invention are administered in combination with one or more additional therapeutic agents, or in a chain. Suitable therapeutic substances include, but are not limited to, cytotoxic or cytostatic substances. Taxol is also a preferred therapeutic substance as a cytotoxic substance.

Cancer therapeutics are those that kill or limit growth of cancer cells, with minimal damage to the host. Thus such agents take advantage of differences from healthy host cells in cancer cell properties (eg metabolism, angiogenesis or the presence of cell surface antigens). Differences in tumor morphology are potent interventional sites: for example, a secondary therapeutic agent may be an antibody, such as an anti-VEGF antibody, useful for slowing the growth rate by slowing angiogenesis inside a tumor mass. Other therapeutic agents include adducts such as granistron HCl, androgen inhibitors such as leuprolide acetate, antibiotics such as doxorubicin, anti-estrogens such as tamoxifen, anti-metabolites such as interferon alpha-2a, and cytotoxicity such as taxol. Substances, enzyme inhibitors such as las farnesyl-transferase inhibitors, immunomodulators such as aldes-leukin, and nitrogen mustard derivatives such as melphalan HCl are applicable, but not limited thereto.

Therapeutic agents that can be combined with EM164 for improved anticancer efficacy include a variety of substances used in tumor research (e.g. docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole, anastrozole, fullvest Fulvestrant, exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, anteide, bevacis Bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine ), Vinorelb ine), estramustine, mitoxantrone, abarelix, zoleronate, streptozocin, rituximab (rituximab) (Rituxan) , Idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab (ivalumomab (ibritumomab ( Zevalin), tositumomab (Bexxar), interferon α-2b, melphalam, bortezomib (Velcade), altretamine, asparagine Asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptor antibody (Cetuximab, Abx-EGF) , And epothilones, and conjugates of antibodies with cytotoxic drugs against cell-surface receptors (see Cancer, Principles & Practice of Oncology, DeVita, VT, Hellman, S., Rosenberg, S. A., 6th edition, Lippincott-Raven, Philadelphia, 2001). Preferred therapeutic agents are platinum materials (eg carboplatin, oxaliplatin, cisplatin), taxanes (eg paclitaxel, docetaxel), gemcitabine, and camptothecin.

One or more additional therapeutic agents may be administered before, simultaneously, or after the antibody, antibody fragment or conjugate of the invention. The skilled artisan will understand that there is a benefit in the particular order of administration for each treatment. Similarly, the skilled person will understand that for each therapeutic agent, the time required between the therapeutic agent and the antibody, antibody fragment or conjugate of the invention will be administered will vary.

The skilled person will understand that the dosage of each therapeutic agent will vary depending on the type of therapeutic agent, with preferred dosages ranging from about 10 mg / m ³ to about 2000 mg / m ³ , more preferably from about 50 mg / m ³ to about 1000 mg / m ³ range is possible. Preferred dosages for preferred therapeutic agents such as platinum materials (carboplatin, oxaliplatin, cisplatin) range from about 10 mg / m ³ to about 400 mg / m ³ , and for taxanes (paclitaxel, docetaxel) the preferred dosage is about 20 mg / m ³ 150 mg / m ³ , the preferred dosage for gemcitabine is about 100 mg / m ³ to about 2000 mg / m ³ , and for camptothecin the preferred dosage is about 50 mg / m ³ to about 350 mg / m ³ . The dosage of this and other therapeutic agents will depend on whether the antibodies, antibody fragments or conjugates of the invention are administered concurrently or serially with the therapeutic agent.

Whether the antibodies, antibody fragments or conjugates of the invention, and one or more additional therapeutic agents are administered together or serially occurs as described for the treatment above. Appropriate pharmaceutically acceptable carriers, diluents, and receptors, when administered together, will allow one of ordinary skill in the art to understand that the particular therapeutic agent will be determined according to simultaneous administration.

When present in water-soluble dosage forms rather than when eluted, antibodies will generally be formulated at a concentration of about 0.1 mg / ml to 100 mg / ml, although various limits of the range are acceptable. The dosage of the antibody or conjugate appropriate for the treatment of the disease depends on the type of disease to be treated, the severity and progression of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, the stage of pretreatment, the clinical history of the patient and the antibody as described above. Response, and the doctor's discretion.

Depending on the type and severity of the disease, for example by one or more independent doses or by continuous dosing, preferably from about 1 mg / m3 to about 2000 mg / m3, more preferably from about 10 mg / m3 to An antibody of about 1000 mg / m 3 is the initial dose for treating the patient. For conditions repeated over several days or more, depending on the condition, treatment is repeated until the desired suppression of disease manifestations occurs. However, other dosage methods may be useful and are not excluded.

The invention also includes kits that include one or more components described herein, instructions for use of the components. In a preferred embodiment the kit of the invention comprises an antibody, antibody fragment or conjugate of the invention, and a therapeutic agent. Instructions for use in the present preferred embodiments include the antibodies, antibody fragments or conjugates of the invention, and instructions for inhibiting the growth of cancer cells using therapeutic agents, and / or patients with cancer, the antibodies, antibody fragments or conjugates of the invention, And instructions for use for treating with the therapeutic agent.

Preferably the antibody used in the kit has the same amino acid sequence as the mouse antibody EM164 by mouse hybridoma EM164 (ATCC accession number PTA-4457), and the antibody is its epitope binding fragment, wherein both the antibody and the fragment are specific And to insulin-like growth factor-I receptor. Antibodies and antibody fragments used in the kits may also be resurfaced versions, humanized versions or modified versions with at least one base mutation, deletion or insertion of an EM164 antibody. Antibodies and antibody fragments of each of these three versions retain the same binding specificities as EM164 antibodies.

Preferably, the therapeutic agent used in the kit is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (traceptzumab (Herceptin) ), Capecitabine, tamoxifen, toremfene, letrozole, anastrozole, fulvestrant, exemestane, gosere Goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin), 5-fluoro Uracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine, Mitoxantrone, Abarelix ( abarelix, Zoledronate, Streptozocin, Rituximab (Rituxanb) (Rituxan), Idarubicin, Busulfan, Chlorambucil, Fludarabine, imatinib, cytarabine, ibritumomab (Zevalin), tositumomab (Bexxar), interferon α- 2b, melphalam, bortezomib (Velcade), altretamine, asparaginase, gefitinib (Iressa), erroni Tip (Tarceva), anti-EGF receptor antibody (Cetuximab, Abx-EGF), and epothilone. More preferably the therapeutic agent is a platinum material (eg carboplatin, oxaliplatin, cisplatin), taxane (eg paclitaxel, docetaxel), gemcitabine, or camptothecin.

The components of the kits of the invention are present in a suitable form for the kit, for example in the form of a solution or eluted powder. It will be understood by those skilled in the art that the concentration or amount of components of a kit will vary depending on the type and intention of using each component in the kit.

Cancers and cells referenced in the kit's instructions include breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial cancer, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma, and rhabdomyosarcoma .

1 shows fluorescence activated cell array (FACS) analysis of specific binding to cells overexpressing the human Y1251F IGF-I receptor or human insulin receptor of purified EM164 antibody.

2 shows the binding titration curves for binding of the EM164 antibody to the biotinylated human IGF-I receptor.

Figure 3 shows inhibition of binding of biotinylated IGF-I to human breast cancer MCF-7 cells by EM164 antibody.

4 shows inhibition of IGF-I promoted autophosphorylation of IGF-I receptor in MCF-7 cells by EM164 antibody.

5 shows the inhibition of IRS-1-phosphorylation promoted by IGF-I in MCF-7 cells by EM164 antibody.

6 shows inhibition of IGF-I promoted signal transduction in SaOS-2 cells by EM164 antibody.

7 shows the effect of EM164 on the growth and survival of MCF-7 cells under different growth conditions as MTT analyzed.

8 shows the effect of EM164 on the growth and survival of MCF-7 cells in the presence of various serum concentrations.

9 shows inhibition of growth and survival of IGF-I and serum promoted NCI-H838 cells by EM164 antibody.

10 shows the therapeutic effect on the growth of Calu-6 lung cancer xenografts in mice with either EM164 antibody, Taxol, or a combination of EM164 and Taxol.

11 shows competition between binding of humanized EM164 antibody (v.1.0) and murine EM164 antibodies.

12 shows the cDNA (SEQ ID NO: 49) and amino acid sequence (SEQ ID NO: 50) of the variable region of the light chain leader and murine anti-IGF-I receptor antibody EM164. The arrow indicates the starting point of structure 1. Three CDR sequences according to Kabat are underlined.

FIG. 13 shows the cDNA (SEQ ID NO: 51) and amino acid sequence (SEQ ID NO: 52) of the variable region of the heavy chain leader and murine anti-IGF-I receptor antibody EM164. The arrow indicates the starting point of structure 1. Three CDR sequences according to Kabat are underlined.

14 shows the light and heavy chain CDR amino acid sequences of the EM164 antibody as determined from the Chothia canonical classification definition. AbM modeling software definitions for heavy chain CDRs are also shown. Light chain: CDR1 as SEQ ID NO: 4, CDR2 as SEQ ID NO: 5, and CDR3 as SEQ ID NO: 6. Heavy chain: CDR1 is SEQ ID NO: 1, CDR2 is SEQ ID NO: 2, and CDR3 is SEQ ID NO: 3. AbM heavy chain: CDR1 is SEQ ID NO: 53, CDR2 is SEQ ID NO: 54, and CDR3 is SEQ ID NO: 55.

FIG. 15 shows the light and heavy chain amino acid sequences for the anti-IGF-I-receptor antibody EM164 aligned in germline sequence for the Cr1 (SEQ ID NO: 56) and J558.c (SEQ ID NO: 57) genes. (-) Indicates sequence homology.

16 shows plasmids forming and expressing recombinant chimeric and humanized EM164 antibodies. A) light chain cloning plasmid, B) heavy chain cloning plasmid, C) mammalian antibody expression plasmid.

FIG. 17 shows the 10 most homologous amino acid sequences of the light chains detected in 127 antibodies with the set of structural files used to predict the surface locus of EM164. em164 LC (SEQ ID NO: 58), 2jel (SEQ ID NO: 59), 2pcp (SEQ ID NO: 60), 1nqb (SEQ ID NO: 61), 1kel (SEQ ID NO: 62), 1hyx (SEQ ID NO: 63), 1igf (SEQ ID NO: 64), 1tet (SEQ ID NO: 65), 1clz (SEQ ID NO: 66), 1bln (SEQ ID NO: 67), 1cly (SEQ ID NO: 68), Consensus (SEQ ID NO: 69).

FIG. 18 shows the 10 most homologous amino acid sequences of the heavy chains detected in 127 antibodies with the set of structural files used to predict the surface site locus of EM164.

em164 HC (SEQ ID NO: 70), 1nqb (SEQ ID NO: 71), 1ngp (SEQ ID NO: 72), 1fbi (SEQ ID NO: 73), 1afv (SEQ ID NO: 74), 1yuh (SEQ ID NO: 75), 1plg (SEQ ID NO: 76), 1d5b (SEQ ID NO: 77), 1ae6 (SEQ ID NO: 78), 1axs (SEQ ID NO: 79), 3hfl (SEQ ID NO: 80), Consensus (SEQ ID NO: 81).

Figure 19 shows the average accessibility for each of the (A) light chain and (B) heavy chain variable region sites from the ten most homologous structures. Numbers indicate Kabat antibody sequence position numbers.

20 shows light chain variable region amino acid sequences for murine EM164 (muEM164) and humanized EM164 (huEM164) antibodies.

21 shows heavy chain variable region amino acid sequences for murine EM164 (muEM164, SEQ ID NO: 87) and humanized EM164 (huEM164, SEQ ID NO: 88) antibodies.

FIG. 22 shows huEM164 v1.0 variable region DNA and amino acid sequences for both the light chain (DNA, SEQ ID NO: 89, amino acid SEQ ID NO: 90) and heavy chain (DNA, SEQ ID NO: 91, amino acid SEQ ID NO: 92).

Figure 23 shows humanized EM164 v1.1 (DNA, SEQ ID NO: 93; amino acid SEQ ID NO: 94), v1.2 (DNA, SEQ ID NO: 95; amino acid SEQ ID NO: 96) and v1.3 (DNA, SEQ ID NO: 97 light chain variable region DNA and amino acid sequence for amino acid sequence number: 98).

24 shows growth and survival inhibition of IGF-I promoted MCF-7 cells by humanized EM164 v1.0 antibody and murine EM164 antibody.

25 shows that EM164 inhibits the cell cycle of MCF-7 cells promoted with IGF-I.

26 shows that EM164 inhibits the anti-apoptotic effect of IGF-I and serum. Treatment with EM164 results in apoptosis cells being killed by an increased amount of cleaved CK18 protein.

27 shows the therapeutic effect on the growth of human BxPC-3 pancreatic cancer xenografts in immunodeficient mice by the combination of EM164 antibody, gemcitabine, or EM164 antibody with gemcitabine.

The present invention has been described with reference to the following examples, which are described solely for the purpose of limiting the invention, but are not intended to be described solely.

Example  One: rat EM164  Antibodies

In a first embodiment, the entire primary amino acid structure and cDNA sequence of the murine antibody of the present invention is disclosed with its binding properties and expression means in recombinant form. In addition, a full disclosure of the antibodies and preparations thereof of the present invention is provided so that those skilled in the art of immunology can produce the antibodies without undue experimentation.

A. Formation of anti- IGF- I receptor monoclonal antibody hybridomas

Because they express a number of IGF-I receptors (10 ⁷ or more per cell), cell lines expressing human IGF-I receptors with the Y1251F mutation have been used for immunization. Y1251F-mutation in the cytoplasmic site of the IGF-I receptor resulted in a decrease in transformation and anti-apoptotic signaling, but did not affect IGF-I binding and IGF-I promoting mitotic signaling (O 'Connor, R. et al., 1997, Mol . Cell . Biol . , 17, 427-435; Miura, M. et al., 1995, J. Biol . Chem . , 270, 22639-22644). The mutation nevertheless did not affect antibody formation because the antibodies of this example, which are identical for both the Y1251F mutant and the wild type receptor, bound onto the extracellular portion of the IGF-I receptor.

Cell lines expressing the human IGF-I receptor with the Y1251F mutation are transmitted from 3T3-like cells of IGF-I-receptor-deficient mice to the Y1251F-mutant human IGF-I-receptor gene along with the puromycin resistance gene Was selected by FACS selection using puromycin (2.5 μg / mL) and high IGF-I receptor expression (Miura, M. et al., 1995, J. Biol . Chem . , 270, 22639-22644). ). Cell lines with high IGF-I receptor expression were also selected using high concentrations of puromycin, such as 25 μg / mL, which are toxic to most cells. Surviving colonies were harvested and colonies showing high IGF-I receptor expression were selected.

Six month old CAF1 / J female mice were immunized intraperitoneally on Day 0 with Y1251F-mutant-human-IGF-I-receptor-overexpressing cells ( ⁵ × 10 ⁵ cells, suspended in 0.2 mL PBS). Mice were expanded with 0.2 mL cell suspension as follows: Day 2, 1 × 10 ⁶ cells; Day 5, 2 × 10 ⁶ cells; 7, 9, 12, and 23rd day, 1 × 10 ⁷ cells. On day 26 mice were sacrificed and their spleens removed.

The spleen was milled between two protruding glass slides to obtain a single cell suspension, which was washed with serum free RPMI medium containing penicillin and streptomycin (SFM). Spleen cell pellets were resuspended for 10 minutes to elute red blood cells in 10 mL of 0.83% (w / v) ammonium chloride solution and then washed with serum free medium (SFM). Splenocytes are filled in tubes with myeloma cells (4x10 ⁷ ) from (1.2x10 ⁸ ) non-vented mouse myeloma cell line P3X63Ag8.653 (ATCC, Rockville, MD; Cat. # CRL1580) and serum-free RPMI-1640 medium Washed with (SFM). Suspension was removed and cell pellet was resuspended in resident medium. The tube was placed in a 37 ° C. water beaker and 1.5 mL of polyethylene glycol solution (50% PEG (w / v), average molecular weight 1500, in 75 mM HEPES, pH 8) was added slowly at a rate of 0.5 mL / min while the tube was mixing. It became. After 1 minute waiting, 10 mL of SFM was added as follows: 1 mL for the first 1 minute, then 2 mL for 1 minute, then 7 mL for 1 minute. Then another 10 mL was added slowly for 1 minute. Cells are pelleted by centrifuge, washed in SFM and 5% bovine serum (FBS), hypoxanthine / aminopterin / thymidine (HAT), penicillin, streptomycin, and 10% hybridoma cloning supplement ( Resuspended in RPMI-1640 growth medium filled with HCS). Cells were seeded by 2 × 10 ⁵ spleen cells in 200 mL per well into a 96-well flat-bottom tissue culture plate. After 5-7 days 100 mL per well was removed and replaced with growth medium filled with hypoxanthine / thymidine (HT) and 5% FBS. General conditions used for immunization and hybridoma production are described in J. Langone and H. Vunakis (Eds., Methods in Enzymology, Vol. 121, "Immunochemical Techniques, Part I";1986; Academic Press, Florida) and E. Harlow and D. Lane ("Antibodies: A Laboratory Manual";1988; Cold Spring Harbor Laboratory Press, New York). Other immunization, hybridoma production techniques can also be used as is well known to those skilled in the art. Culture suspended solids from hybridoma clones were cloned for binding to human IGF-I receptor purified by ELISA, for specific binding to cells overexpressing human IGF-I receptor, and by ELISA and FACS screening. Likewise detected for the absence of binding to cells overexpressing the human insulin receptor. Clones that showed higher binding affinity for cells overexpressing the human IGF-I receptor than for cells overexpressing the human insulin receptor were expanded and subcloned. Culture suspensions of subclones were also detected by the binding assay. Subclone 3F1-C8-D7 (EM164) was selected by this process and the heavy and light chain genes were cloned and sequenced as follows.

Human IGF-I receptors were isolated from hybridoma clones by the following method for their binding to the IGF-I receptor for use in the detection of floats. Biotinylated IGF-I was prepared using a biotinylation reagent such as sulfo-NHS-LC-biotin, sulfo-NHS-SS-biotin, or NHS-PEO ₄ -biotin by modification of recombinant IGF-I. . Biotinylated IGF-I was incubated with lysates from cells that were absorbed on streptavidin-agarose beads and overexpressed human wild type or Y1251F mutant IGFR. Beads were washed and removed with buffer containing detergents such as 2-4 M urea and Triton X-100 or octyl-b-glucoside. The removed IGF-I receptor was dialyzed against PBS and analyzed by SDS-PAGE under reducing conditions for purification, showing the alpha and beta chains of the IGF-I receptor with molecular weights of 135 kDa and 95 kDa, respectively. gave.

To confirm binding of the hybridoma suspension to the purified IGF-I receptor,

Immulon-4HB ELISA plate (Dynatech) was coated with purified human IGF-I receptor samples diluted (100 ml; 4 ° C., overnight) in pH9.5 50 mM CHES buffer (urea / octyl- of affinity purified samples). prepared by dialysis from b-glucoside removal). Wells were blocked with 200 ml of blocking buffer (10 mg / mL BSA TBS-T buffer containing 50 mm TRIS, 150 mm NaCl, pH 7.5, and 0.1% TWEEN-20) and with suspension from hybridoma clones. Incubated for 1-12 hours (100 mL; whitened in blocking buffer), washed with TBS-T buffer, incubated with goat-anti-mouse-IgF-Fc-antibody-horseradish peroxidase (HRP) conjugate ( 100 ml; 0.8 mg / ml in blocking buffer; Jackson Immuno Research Laboratory), wash and ABTS / H ₂ O ₂ at 405 nm (0.5 mg / mL ABTS, 0.03% H ₂ O _{2 in} 0.1 M citric acid buffer, pH 4.2) Used Observation followed. Typically, the suspension from the 3F1 hybridoma subclone produced a signal with absorbance of about 1.2 during three minutes of expression, in contrast to the value of 0.0 obtained from the suspension from some other hybridoma clones. General conditions for this ELISA include standard ELISA conditions for antibody binding and detection as described by E. Harlow and D. Lane ("Using Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor Laboratory Press, New York). Similar.

Specific binding to human IGF-I receptors and detection of hybridoma suspensions that do not bind to human insulin receptors results in ELISA on cell lines overexpressing the human Y1251F-IGF-I receptor and cell lines overexpressing the human insulin receptor. It was carried out using. Both cell lines were generated from 3T3-like cells of IGF-I receptor deficient mice. IGF-I receptors overexpressing cells and insulin receptors overexpressing cells are isolated, grown by rapid trypsin / EDTA treatment from tissue culture flasks, suspended in growth medium containing 10% FBS and pelleted by centrifuge And washed with PBS. Washed cells (100 mL of about 1-3 × 10 ⁶ cells / mL) are added to the wells of Immulon-2HB plates coated with phytohemagglutinin (100 mL of 20 mg / mL PHA), centrifuged and , Adhered to PHA-coated wells for 10 minutes. Plates with cells were fried to remove PBS and then dried at 37 ° C. overnight. Wells were blocked with 5 mg / mL BSA solution in PBS for 1 hour at 37 ° C. and then washed with PBS. Suspensions from hybridoma clones (100 mL; diluted in blocking buffer) were then added to wells containing IGF-I-receptor-overexpressing cells and wells containing insulin receptor-overexpressing cells and at ambient temperature. Incubated for 1 hour at. Wells were washed with PBS and incubated for 1 hour with goat-anti-mouse-IgG-Fc-antibody-horseradish peroxidase conjugate (100 mL; 0.8 mg / mL in blocking buffer) followed by washing followed by ABTS / H ₂ O Binding was detected using ₂ . Typical suspensions from 3F1 hybridoma subclone when cultured with cells overexpressing IGF-I receptor produced 0.88 absorbance signals during 12 min expression, with 0.22 absorbance values during cell culture overexpressing human insulin receptor. In contrast to that. Hybridomas were grown in Integra CL 350 flasks (Integra Biosciences, Maryland) to produce purified EM164 antibodies. Suspensions grown from Integra flasks yielded about 0.5-1 mg / mL antibody production based on quantification by ELISA and SDS-PAGE / Coomassie blue staining. Antibodies were purified by affinity chromatography on a Protein A-agarose bead column under standard purification conditions leading to the discharge of 100 mM Tris buffer, pH 8.9, containing 3M NaCl, containing 100 mM acetic acid containing 150 mM NaCl. Removed fragments containing the antibody were neutralized with 2 MK ₂ HPO ₄ solution and dialyzed in 4 ° C. PBS. The concentration of the antibody was determined by measuring absorbance at 280 nm (absorption coefficient = 1.4 mg ^-1 mL cm ^-1 ). Purified antibody samples were analyzed by reducing conditions SDS-PAGE and Coomassie Blue staining, which showed only heavy and light chain bands at 55 kDa and 25 kDa, respectively. The isotype of the purified antibody was IgG ₁ with κ light chain.

B. Binding Characterization of EM164 Antibodies

Specific binding of the purified EM164 antibody showed fluorescence activated cell sorting (FACS) using cells overexpressing the human IGF-I receptor and cells overexpressing the human insulin receptor (FIG. 1). Incubation of EM164 antibody (50-100 nM) in 100 mL cold FACS buffer (1 mg / mL BSA in Dulbecco's MEM medium) resulted in cells overexpressing human IGF-I receptor and cells overexpressing human insulin receptor (2x10). ⁵ cells / mL). Cells were pelleted by centrifuge and washed with cold FACS buffer and incubated with goat-anti-mouse-IgG-antibody-FITC conjugate for 1 hour on ice (100 mL; 10 mg / mL in FACS buffer). Cells were resuspended in pellet, washed, 120 mL of 1% formaldehyde solution in PBS. Plates were analyzed using a FACSCalibur reader (BD Biosciences).

Strong fluorescence shift was obtained in culture of IGF-I receptors overexpressing cells with EM164 antibody, in contrast to small shifts in culture of insulin receptors overexpressing cells with EM164 antibody (FIG. 1). It was described that binding to the IGF-I receptor is selective and not to the insulin receptor. Control antibody, anti-IGF-I receptor antibody 1H7 (Santa Cruz Biotechnology) and anti-insulin alpha antibody (BD Pharmingen Laboratories) produced fluorescence shift in culture with cells overexpressing IGF-I receptor and insulin receptor (FIG. One). Strong fluorescence shift was also observed by FACS analysis using EM164 antibody and human breast cancer MCF-7 cells, which expressed IGF-I receptors (Dufourny, B. et al., 1997, J. Biol . Chem . , 272) . , 31163-31171), showed EM164 antibodies bound to the IGF-I receptor on the surface of human human tumor cells.

The isolation constant ( K _d ) for binding EM164 antibody to human IGF-I receptor was determined by ELISA titration of antibody binding at various concentrations, either directly coated IGF-I receptor or indirectly captured biotinylated IGF-I receptor. Determined by Biotinylated IGF-I receptors were prepared by biotinylation of detergent-stabilized lysates from IGF-I receptors overexpressing cells using PEO-maleimido-biotin reagent (Pierce, Molecular Biosciences), NHS Affinity purified using anti-IGF-I receptor beta chain antibody immobilized on agarose beads or removed with 2-4 M urea buffer containing NP-40 detergent and dialyzed in PBS.

Kd determination of binding EM164 antibody to biotinylated IGF-I receptor was performed by coating Immulon-2HB plates in carbonate buffer with 1 mg / ml 100 mL streptavidin overnight at 4 ° C. (150 mm sodium carbonate, 350 mm Sodium bicarbonate). Streptavidin-coated wells were blocked with 200 mL blocking buffer (10 mg / ml BSA in TBS-T buffer), washed with TBS-T buffer and incubated at ambient temperature for 4 hours with a biotinylated aqueous IGF-I solution. (10 ng to 100 ng). Wells containing indirectly captured biotinylated IGF-I receptors were washed and incubated for 2 hours at ambient temperature with EM164 antibody in blocking buffer at various concentrations (5.1x10 ^-13 M to 200 nM) overnight at 4 ° C. Incubated.

Wells were then washed with TBS-T buffer and incubated with goat-anti-mouse-IgG _{H + L} -antibody-horseradish peroxidase conjugate (100 mL; 0.5 mg / mL in blocking buffer) and washed and ABTS / at 405 nm. Detection with H ₂ O ₂ was followed. K _d values were determined by non-linear decrease in single-site binding.

Similar binding titrations were performed using Fab fragments of EM164 antibody and papain as described by E. Harlow and D. Lane ("Using Antibodies: A Laboratory Manual"; 1999, Cold Spring Harbor Laboratory Press, New York). Prepared by absorption.

Binding titration curves for the biotinylated human IGF-I receptor of the EM164 antibody produced 0.1 nM K _d values (FIG. 2). Fab fragments of the EM164 antibody also bound the human IGF-I receptor very tightly with a K _d value of 0.3 nM, suggesting that the monomer binding of the EM164 antibody to the IGF-I receptor is also very potent.

The very low value of the separation constant for the binding of the IGF-I receptor by this EM164 antibody is partly as evidenced by the strong binding signal observed after 1-2 days wash of the antibody bound to the immobilized IGF-I receptor. This is due to the very slow k _off speed.

The EM164 antibody was immunoprecipitated of the IGF-I receptor as described by culturing detergent-stabilizing lysates of human breast cancer MCF-7 cells with EM164 antibody immobilized on protein G-Agarose beads (Pierce Chemical Company). Can be used for

Western blots of EM164 antibody immunoprecipitates were detected using rabbit polyclonal anti-IGF-I receptor beta chain antibody (C-terminus) (Santa Cruz Biotechnology) and goat-anti-rabbit-IgG-antibody-horseradish peroxidase conjugates Followed by washing, and increased luminescence (ECL) detection. Western blots of EM164 immunoprecipitates from MCF-7 cells showed bands corresponding to beta chains of IGF-I receptor 95 kDa and pro-IGF-I receptor about 220 kDa. Similar immunoprecipitation occurred for other cell types to confirm species specificity of the binding of EM164 antibody and also bound to IGF-I receptors from cos-7 cells (African green monkeys), but 3T3 cells (mouse), CHO It did not bind to the IGF-I receptor of cells (Chinese hamster) or goat fibroblast cells (goat). EM164 antibody did not detect SDS-denatured human IGF-I receptors in western blot of lysates from MCF-7 cells, showing binding to epitopes consistent with the original, undenatured human IGF-I receptors gave. The binding domain of the EM164 antibody was also specified using a truncated alpha chain, rich in cysteine (1-468 sites), where the L1 and L2 domains were flanked by fusion to the 16-mer-C-terminal (704-719 sites). Domain was included and terminated with C-terminal epitope selection. This small IGF-I receptor lacks the 469-703 site and has been recorded to bind IGF-I (Molina, L. et al., 2000, FEBS Letters , 467, 226-230; Kristensen, C.). et al., 1999, J. Biol . Chem . , 274, 37251-37356). Thus, the truncated IGF-I receptor alpha chain was prepared containing 704-719 sites and 1-468 sites fused to the C-terminal side flanked by C-terminal myc epitope selection. A fixed cell line was formed that expresses this construct and also transiently expresses this construct in human embryonic kidney 293T cells. Strong binding to the cleaved IGF-I receptor alpha chain construct of the EM164 antibody was detected. Of the two antibodies, IR3 (Calbiochem) was also bound to this cleaved alpha chain, but did not bind to the 1H7 antibody (Santa Cruz Biotechnology), which showed that the epitope of the EM164 antibody was clearly distinguished from the 1H7 antibody.

C. Inhibition of IGF- I Binding to MCF- 7 Cells by EM164 Antibody

Binding of IGF-I to human breast cancer MCF-7 cells was inhibited by EM164 antibody (FIG. 3). MCF-7 cells were cultured in serum-free medium for 2 hours with or without 5 mg / mL EM164 and followed by incubation at 37 ° C. for 20 minutes with 50 ng / mL biotinylated IGF-I. Cells were washed twice to remove unbound Biotin-IGF-I with serum free medium and then eluted in 50 mM HEPES, pH 7.4, containing 1% NP-40 and protease inhibitors. Immulon-2HB ELISA plates were coated with mouse monoclonal anti-IGF-I receptor beta chain antibodies and used to capture IGF-I receptors and bound biotin-IGF-I from the lysate. Binding of the coated antibody to the beta chain cytoplasmic C-terminal domain of the IGF-I receptor did not interfere with the binding of the extracellular domain of biotin-IGF-I. Wells were washed and incubated with streptavidin-horseradish peroxidase conjugate, rewashed, and detected using ABTS / H ₂ O ₂ substrate. Inhibition of IGF-I binding to MCF-7 cells by the 5 mg / mL EM164 antibody was necessarily quantitative and nearly identical to that of the ELISA background obtained using no control biotin-IGF-I.

In addition to the experiments described above for the inhibition of binding of IGF-I to MCF-7 cells by the EM164 antibody, the following assay is performed by an endogenous physiological ligand (e.g., IGF-) bound to an anti-IGF-I receptor antibody. It was demonstrated that the EM164 antibody-bound IGF-I from MCF-7 cells was highly effective under physiological conditions for replacing I or IGF-II). In this IGF-I replacement experiment, MCF-7 cells grown in 12-well plates were depleted in serum and biotinylated IGF-I (20- for 1 or 2 hours at 37 ° C. (or 4 ° C.) in serum-free medium. 50 ng / mL). Cells with bound biotinylated IGF-I were then treated with EM164 antibody or control antibody (10-100 mg / mL) at 37 ° C. (or 4 ° C.) for 30 minutes to 4 hours. The cells were then washed with PBS and eluted in elution buffer containing 1% NP-40 at 4 ° C. ELISA was performed to capture the IGF-I receptor from the lysate and then detect the biotinylated IGF-I bound to the receptor using a streptavidin-horseradish peroxidase conjugate as described above. This ELISA showed biotinylated IGF-I pre-bound with EM164 antibody to cells at 37 ° C. almost completely (90% in 30 minutes and ˜100% in 4 hours) and at 4 ° C. over 2 hours. It was shown that about 50% replaceable. In another example, NCI-H838 lung cancer cells were incubated with Biotin-IGF-I, washed and incubated for 2 hours at 4 ° C. with EM164 antibody, resulting in an 80% reduction in bound Biotin-IGF-I. Brought it. Thus, the EM164 antibody was very effective in replacing prebound IGF-I from cancer cells and would be of therapeutic importance in the hyperactivity of the IGF-I receptor by replacement of the bound endogenous physiological ligand.

Although longer cultures with EM164 resulted in 25% downregulation of the IGF-I receptor, incubating MCF-7 cells with EM164 antibodies for 2 hours at 4 ° C. (or 30 minutes at 37 ° C.) was performed in Western blot analysis. Based on anti-IGF-I receptor beta chain antibodies (Santa Cruz Biotechnology; sc-713) did not result in significant downregulation of the IGF-I receptor. Therefore, the inhibition of IGF-I binding and replacement of bound IGF-I by EM164 antibody in these short-term experiments at both 4 ° C. and 37 ° C. should not be explained by downregulation of receptors due to binding of EM164 antibody. The mechanisms for the strong inhibition of binding of IGF-I to the IGF-I receptor and the replacement of prebound IGF-I by EM164 antibodies are likely to be binding competition through allocation of binding sites or steric hindrance or other site effects. .

D. EM164 By antibody IGF -I Receptor-mediated Cell Signaling

 Treatment of breast cancer MCF-7 cells and osteosarcoma SaOS-2 cells with EM164 antibody inhibits IGF-I receptor autophosphorylation and phosphorylation of downregulatory effectors such as insulin receptor substrate-1 (IRS-1), Akt and Erk1 / 2 As shown by inhibition, intracellular IGF-I receptor signaling was almost completely inhibited (FIGS. 4-6).

In FIG. 4, MCF-7 cells were grown for 3 days in standard medium in 12-well plates and then treated with 20 mg / mL EM164 antibody (or anti-B4 control antibody) for 3 hours in serum-free medium and 50 ng / Stimulation was continued at 37 ° C. for 20 minutes with mL IGF-I. The cells were then ice-cold elution buffer (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium) containing protease and phosphatase inhibitors. Eluted in pyrophosphate, 2.5 mM EDTA, 10 mM leupetin, 5 mM pepstatin, 1 mM PMSF, 5 mM benzamidine, and 5 mg / mL aprotinin).

ELISA plates were pre-coated with anti-IGF-I receptor beta chain C-terminal monoclonal antibody TC123 and incubated at ambient temperature for 5 hours with lysate samples to capture IGF-I receptor. Wells containing captured IGF-I receptors are then incubated for 30 minutes with washed and biotinylated anti-phosphotyrosine antibody (PY20; 0.25 mg / mL; BD Transduction Laboratories), washed and streptavidin-horseradish fur Incubation was continued for 30 minutes with oxidase conjugate (0.8 mg / mL).

Wells were washed and detected with ABTS / H ₂ O ₂ substrate. The use of control anti-B4 antibodies showed no inhibition of IGF-I promoting autophosphorylation of the IGF-I receptor. In contrast, complete inhibition of IGF-I promoting autophosphorylation of the IGF-I receptor was obtained upon EM164 antibody treatment (FIG. 4).

ELISA with immobilized anti-IRS-1 antibody was used to capture IRS-1 from lysate to demonstrate the inhibition of phosphorylation of insulin receptor substrate-1 (IRS-1) and bind to phosphorylated IRS-1 A subsequent p85 subunit measurement of phosphatidylinositol-3-kinase (PI-3-kinase) was followed (Jackson, JG et al., 1998, J. Biol . Chem . , 273, 9994-10003). In FIG. 5 MCF-7 cells were treated with 5 mg / Ml antibody (EM164 or IR3) for 2 hours in serum-free medium followed by stimulation at 37 ° C. for 10 minutes with 50 ng / mL IGF-I. Anti-IRS-1 antibody (Rabbit Polyclonal; Upstate Biotechnology) was captured by incubation on ELISA plates with indirectly coated goat-anti-rabbit-IgG antibodies, and plates were removed from cell lysates by incubation overnight at 4 ° C. It was used to capture IRS-1. Wells were then incubated for 4 hours with mouse monoclonal anti-p85-PI-3-kinase antibody (Upstate Biotechnology) followed by 30 minutes treatment with goat-anti-mouse- IgG-antibody-HRP conjugate. Wells were then washed and detected using ABTS / H ₂ O ₂ substrate (FIG. 5). As shown in FIG. 5, the EM164 antibody was more effective than the IR3 antibody in inhibiting IGF-I-promoting IRS-1 phosphorylation, and the EM164 antibody had no anti-inflammatory action in IRS-1 phosphorylation when cultured with cells lacking IGF-I. Not shown.

Activation of other downdraft effectors, such as, for example, Akt and Erk1 / 2, is characterized by Western blot and phosphate-specific antibodies (rabbit polyclonal anti-phospho-Ser ⁴⁷³ Akt polyclonal and anti-phospho-ERK1 / 2 antibodies; Cell Signaling Technology) was also inhibited by EM164 antibodies in the SaOS-2 gene (Figure 6) and MCF-7 cells in a dose-dependent manner. pan-ERK antibodies showed the same protein loading in all lanes (FIG. 6). Treatment of SaOS-2 cells with EM164 did not inhibit EGF-promoting phosphorylation of Erk1 / 2 and thus did not show the specificity of inhibiting the IGF-I receptor signaling pathway.

E. Inhibition of IGF- I-, IGF - II- and Serum-Promoted Growth and Survival of Human Tumor Cells by Antibodies

Various human tumor cells have been tested in serum-free conditions for their growth and survival response to IGF-I. These cell lines were treated with EM164 antibody in the presence of IGF-I, IGF-II, or serum, and their growth and survival responses were measured by MTT assay after 2-4 days. About 1500 cells were plated in 96-well plates in regular medium with serum and replaced with serum-free medium the next day (serum-free RPMI medium was filled with transferrin and BSA or Dufourny, B. et al., 1997, J ... Biol Chem, 272, 31163-31171, as specified by the non-phenol red medium Im). After one day of growth in serum-free medium, cells are incubated with 75 ml of 10 mg / ml antibody for 30 minutes.-3 hours, with a final concentration of 10 ng / mL IGF-I, or 20 ng / mL IGF-II, or 0.04. This was followed by the addition of a 25 ml solution of IGF-I (or IGF-II or serum) to obtain -10% serum. In some embodiments, cells were first stimulated with IGF-I for 15 minutes prior to addition of EM164 antibody, or both IGF-I and EM164 antibody were added together. Then the cells were allowed to grow for another 2-3 days. MTT (3- (4,5) -dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; 25 ml of a 5 mg / ml solution in PBS) was then added, and the cells were returned to the incubator for 2-3 hours. The medium was then removed and replaced with 100 ml DMSO, mixed, and the absorbance of the plate was 545 Measured at nm. Regardless of whether the antibody was added before IGF-I, or whether IGF-I and the antibody were added together, various human tumor cells significantly increased upon addition of IGF-I or IGF-II or serum inhibited by EM164 antibody. Growth and survival responses were shown (Table 1).

Table 1. EM164  By antibody IGF Inhibition of Growth and Survival of -I-Promoted Tumor Cells

Tumor cell types Fold growth response to IGF-I (MTT assay: ratio of treated IGF-I to untreated cells in serum-free medium) ^a % Inhibition of IGF-I-stimulatory growth by EM164 antibody in serum-free medium Inhibition by EM164 Antibody of IGF-I-stimulatory Growth / Survival in 1.25-10% Serum ^b MCF-7 (Breast) 1.7-2.8 100% 85% HT-3 (cervical neck) 2 70-90% ND Colo 205 (Colon) 2.3 50% Yes HT-29 1.5 60% Yes NCI-H838 (Lung) 3 100% 85-90% Calu-6 1.6-1.8 85% Yes SK-LU-1 1.4 100% No NCI-H596 1.4 100% Weakly A 549 1.2 80% ND A 375 (Melanoma) 1.6 90% No SK-Mel-37 1.4 85% ND RD (Rhabdomyosarcoma) 1.7 85-100% Yes SaOS-2 (osteosarcoma) 2.5 100% Yes A 431 (epithelial) 2.2 85% Yes SK-N-SH (neuroblastoma) 2 85% 30-50%

( ^a MTT assay for 3- to 4-day cell growth / survival in response to 10 ng / mL IGF-I in serum-free medium containing 5-10 mg / mL EM164 antibody)

colonization formation based on comparison with MTT assay or control (including serum but no antibody) for inhibition of cell growth in the presence of 5-10 mg / mL EM164 antibody in ^b 1.25-10% serum; To explain lateral IGF-stimulation, the extent of inhibition was measured quantitatively for MCF-7, NCI-H838, and SK-N-SH cells based on controls (without serum but with antibodies and with serum but without antibodies) ND indicates no data or poor data due to difficulty in dyeing)

EM164 antibody strongly inhibited IGF-I- or serum-stimulatory growth and survival of breast cancer MCF-7 cells (FIGS. 7 and 8). In an isolated example, the EM164 antibody strongly inhibited IGF-II-stimulated growth and survival of MCF-7 cells. Previous reports using commercial antibodies such as IR3 antibodies showed weak inhibition of serum-stimulated growth and survival of only MCF-7 cells as confirmed in FIG. 7 for IR3 and 1H7 antibodies (Cullen, KJ et al., 1990, Cancer Res . , 50, 48-53). In contrast, EM164 antibodies were potent inhibitors of growth IGF-stimulation of serum or MCF-7 cells. As shown in FIG. 8, EM164 antibody was equally effective in inhibiting growth and survival of MCF-7 cells in the serum concentration range (0.04-10% serum).

Growth inhibition of MCF-7 cells by EM164 antibody was measured by counting the cells. Thus, in 12-well plates, about 7500 cells were plated in 10% FBS medium in the presence or absence of 10 mg / ml EM164 antibody. After 5 days of growth, the cells were counted 20.5 × 10 ⁴ cells for untreated control specimens, whereas only 1.7 × 10 ⁴ cells were counted for EM164 antibody treated specimens. Five days after treatment with EM164 antibody, the growth of MCF-7 cells was inhibited by about 12-fold. Inhibition by EM164 antibody increased more than 2.5-fold inhibition using IR3 antibody 6-days after analysis on MCF-7 cells (Rohlik, QT et al., 1987, Biochem . Biophys . Res) . Commun., 149, 276-281) .

IGF-I- and serum stimulatory growth and survival of non-small cell lung cancer line NCI-H838 was strongly inhibited by EM164 antibody (FIG. 9). Treatment with EM164 antibody in serum-free medium produced smaller signals than if untreated for both NCI-H838 and MCF-7 cells, because EM164 antibody also had autologous and lateral IGF-I and IGF-II of these cells. This is because the stimulation is suppressed (FIGS. 7 and 9). The colony size of HT29 colon cancer cells was also significantly reduced when treated with EM164 antibody.

Thus, EM164 antibody is specific among all anti-IGF-I receptor antibodies known for their serum stimulatory growth inhibition efficiency of tumor cells such as MCF-7 cells and NCI-H838 cells. EM164 antibody caused growth arrest of cells in the G0 / G1 phase and stopped the mitotic effect of IGF-I. For cell cycle analysis, MCF-7 cells were treated with IGF-I (20 ng / ml) for 1 day in the presence or absence of EM164 (20 mg / ml), and by propidium iodide staining and flow cytometry Analyzed. As shown in FIG. 25, the cell cycle was suppressed in EM164-treated cells (41% cells in phase S and 50% in phase G0 / G1) in response to IGF-I stimulation in the absence of EM164 (S phase S). 9% cells and 77% of the G0 / G1 phase).

In addition to inhibition of cell proliferation, EM164 antibody treatment resulted in apoptosis of cells. For the measurement of apoptosis, cleavage of the cytokeratin CK18 protein by caspase was carried out in NCI-H838 lung cancer cells cultured with IGF-I or serum with or without IGF-I or serous for 1 day. It was measured (Figure 26). In the absence of EM164, addition of IGF-I or serum resulted in lower caspase-cleaved CK18 signal, which meant that IGF-I and serum inhibited the activation of caspase. Treatment with EM164 inhibited the anti-apoptotic effect of IGF-I and serum, which was manifested by larger cleaved CK18 obtained in the presence rather than absence of EM164 (FIG. 26).

F. Inhibition of integration by EM164 antibodies of human tumor cell growth and survival in combination with other cytotoxic and cytostatic substances

The combined administration of EM164 antibody and Taxol had a significant inhibitory effect on the growth and survival of non-small cell lung cancer Calu6 cells than when Taxol alone was administered. Similarly, the combination of EM164 antibody and camptothecin had a much more inhibitory effect on the growth and survival of colon cancer HT29 cells than when camptothecin alone. Since the EM164 antibody alone was not expected to be as toxic to the cells as the organic chemotoxin, the integration between the cell-fixing function of the EM164 antibody and the cytotoxic function of the chemotoxin is very effective in the treatment of clinical stage complex cancer.

The combined function of the EM164 antibody and the anti-EGF receptor antibody (KS77) is that the growth and survival of various tumor cell lines, such as HT-3 cells, RD cells, MCF-7 cells, A431 cells, etc., than the EM164 antibody or KS77 antibody alone. Had a significant inhibitory effect.

Thus, the integrated action of neutralizing antibodies to two growth factor receptors, the IGF-I receptor and the EGF receptor, is useful in the treatment of clinical cancer.

As shown in Table 1, further efficacy studies were conducted using a combination of EM164 antibodies and other anticancer therapies based on the efficacy of EM164 antibodies as the sole agent that inhibits the proliferation and survival of various human cancer cell lines. For these various cancer cell lines in these studies, the combined treatment of EM164 antibodies with other anticancer agents has resulted in much greater anticancer efficacy than with EM164 or other agents alone. These combinations of EM164 and other therapeutic agents are therefore very effective in inhibiting cancer cell proliferation and survival. Therapeutic agents that can be combined with EM164 for improved anticancer efficacy include docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (herceptin (trastuzumab (Herceptin)), capecitabine, tamoxifen, toremfene, letrozole, anastrozole, fulvestrant, exemestane (exemestane), goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin) ), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estorabin Estramustine, mitoxantrone xantrone, abarelix, zoleronate, streptozocin, rituximab (rituximab), rituxan, idarubicin, busulfan, Chlorambucil, fludarabine, imatinib, cytarabine, cytarabine, ibritumomab (Zevalin), tositumomab (tositumomab ( Bexxar), interferon α-2b, melphalam, bortezomib (Velcade), altretamine, asparaginase, zefitinib (gefitinib) (Iressa), erlonitib (Tarceva), anti-EGF receptor antibodies (Cetuximab, Abx-EGF), and cytotoxins against epothilones and cell surface receptors These include various drugs used in tumor procedures such as conjugates of antibodies ( Reference : Cancer, Principles & Practice of Oncology, DeVita, VT, Hellman, S., Rosenberg, S A, 6th edition, Lippincott-Raven, Philadelphia, 2001).

For these combination therapies, EM164 is a combination of alkylating agents, platinum substances, hormonal therapies, antimetabolic agents, topoisomer inhibitors, antimicrotubule substances, differentiators, anti-angiangiogenic or anti-conduit therapies, radiotherapy, LHRH or GnRH antagonists and agonists, cells inhibited by against the pomyeon receptor antibodies or small molecule inhibitors, and are combined with one or more anticancer drugs with different active mechanisms, such as other chemotherapeutic agents (Reference: cancer, Principles & Practice of Oncology, DeVita, VT, Hellman, S., Rosenberg, SA, 6th edition, Lippincott-Raven, Philadelphia, 2001). In one embodiment, the combination of LHRH antagonist antide (0.1-10 μM) and EM164 antibody (0.1-10 nM) significantly inhibited the spread of MCF-7 breast cancer cells than when treated with EM164 or antide alone.

In combination treatment with platinum material, the combined treatment of EM164 antibody (10 μg / ml) and cisplatin (0.1-60 μg / ml) resulted in greater proliferation and survival of MCF-7 breast cancer cells than EM164 or cisplatin alone. It has an inhibitory effect.

Combination of EM164 antibodies with other therapeutic agents is effective against various types of cancers including breast cancer, colon cancer, lung cancer, prostate cancer, pancreatic cancer, cervical cancer, ovarian cancer, melanoma cancer, multipolar myeloma cancer, neuroblastoma, rhabdomyosarcoma and osteosarcoma . The EM164 antibody and therapeutic agent may be administered simultaneously or sequentially to the cancer treatment.

Conjugates of the EM164 antibody and cytotoxin are also valuable for targeted delivery of tumors that overexpress the IGF-I receptor of the cytotoxin. Conjugates of EM164 antibodies and radiolabels or other labels can be used in tumor therapy and imaging that overexpress IGF-I receptors.

G. Effect of EM164 treatment on human cancer xenografts in immunodeficient mice in the form of a single agent or anticancer agent

Human non-small cell lung cancer Calu-6 xenografts were formed in immunodeficient mice by subcutaneous injection of 1 × 10 ⁷ Calu-6 cells. As shown in FIG. 10, these mice comprising 100 mm ³ Calu-6 xenografts formed were treated with EM164 antibody alone (6 injections of 0.8 mg / mouse, iv, twice per week) using 5 mice per treatment group, Taxol alone (5 injections of Taxol, every 2 days; 15 mg / kg), or a combination of Taxol and EM164, or PBS alone (200 mL / mouse, 6 injections, twice a week, iv). Tumor growth was significantly delayed by EM164 antibody treatment when compared to PBS controls. Non-toxicity of EM164 antibody was observed based on mouse weight measurement. Although Taxol alone was effective until 14 days, tumors began to grow again. However, tumor growth in the group treated with the combination of Taxol and EM164 antibodies was significantly delayed compared to the group treated with Taxol alone.

Human pancreatic cancer xenografts were formed by subcutaneous injection of 10 ⁷ BxPC-3 cells in PBS in 5 week old female SCID / ICR mice (Taconic) (day 0). Mice producing 80 mm ³ tumors formed were then treated with EM164 alone (0.8 mg / mouse 13 injections, iv, lateral tail veins, 12, 16, 19, 23, 26, 29, 36, 43, 50, 54, 58 , Day 61, 64), gemcitabine alone (2 injections of 150 mg / kg / mouse, ip, 12 and 19), with gemcitabine and EM164 in combination, PBS alone, and controls according to the following steps Antibodies alone (following the same progression steps as EM164) were treated with 5 mice each out of 5 treatment groups. As shown in FIG. 27, treatment with EM164 alone or in combination with gemcitabine initially resulted in a total reduction in tumor xenografts in four of five in the EM164 treatment group and in five in the combination treatment group. Measurable tumor regrowth was seen only in one or more animals at Day 43 in the EM164 group and Day 68 in the combination treatment group, 74 compared to the control treatment (P = 0.029 and 0.002, respectively; 2-tailed T -test; FIG. 27). The primary resulted in smaller microtumor volumes.

On another date, EM164 antibody treatment (alone or in combination with anti-EGF receptor antibodies; intraperitoneal injection) inhibited the growth of formed mouse BxPC-3 xenografts.

Murine EM164 and humanized EM164 antibodies showed the same inhibition of BxPC-3 xenograft growth in the mice formed, thus showing that the intensity of humanized EM164 in vivo is comparable to that of the rat EM164. When comparing different forms of EM164 antibody administration, both intraperitoneal and vascular administration of EM164 antibody showed comparable inhibition of the growth of BxPC-3 xenografts in mice formed. In another xenograft study, EM164 antibody treatment showed significant growth retardation of A-673 human rhabdomyosarcoma / Ewing's tumor xenografts in formed mice.

H. Light and Heavy Chains of EM164 Antibody Cloning and Sequencing

Total RNA was purified from EM164 hybridoma cells. Reverse transcriptase reactions were performed using 4-5 μg total RNA and oligo dT or random hexamer primers.

PCR reactions were performed by Co et al. Using the RACE method described in (J. Immunol., 148, 1149-1154 (1992)) and the degradation primers described in Wang et al., (J. Immunol. Methods, 233, 167-177 (2000)). Was carried out. The RACE PCR method required an intermediate step to add a poly G tail at the 3 'end of the first helix cDNA. RT reaction was purified by Qianeasy (Qiagen) column and removed in 50 μl 1 × NEB buffer 4. dG tailing reaction was performed with 0.25 mM CoCl ₂ , 1 mM dGTP, and 5-unit terminal transferase (NEB) (in 1 × NEB buffer 4). The mixture was incubated at 37 ° C. for 30 minutes and then one fifth of the reaction (10 μl) was added directly to the PCR reaction to act as a DNA fragment.

RACE and digestion PCR reactions were identical except for primer and fragment differences. Terminal transferase reactions were used directly for RACE PCR fragments, and RT reaction mixtures were used directly for digestion PCR reactions.

Identical 3 'light chain primers in RACE and digestion PCR reactions: HindKL-tatagagctcaagcttggatggtgggaagatggatacagttggtgc (SEQ ID NO: 14)

And 3 'heavy chain primers:

Bgl2IgG1-ggaagatctatagacagatgggggtgtcgttttggc (SEQ ID NO: 15)

Was used.

In RACE PCR, one poly C 5 ′ primer was used for both heavy and light chains:

EcoPolyC-TATATCTAGAATTCCCCCCCCCCCCCCCCC (SEQ ID NO: 16),

The cleaved 5 'terminal PCR primers are as follows:

For light chains Sac1MK-GGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 17), and the same mix of:

EcoR1MH1-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO: 18) and

For heavy chains EcoR1MH2-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO: 19).

In the primer sequence, the mixed base is defined as follows: H = A + T + C, S = g + C, Y = C + T, K = G + T, M = A + C, R = A + g, W = A + T, V = A + C + G.

The PCR reaction was carried out using the following program: 1) 94 ° C. 3 minutes, 2) 94 ° C. 15 seconds, 3) 45 ° C. 1 minute, 4) 72 ° C. 2 minutes, 5) Return to Step # 2 Cycle 29 6) terminated with a final extension step at 72 ° C. for 10 minutes.

PCR products were cloned into pBluescript II SK + (Stratagene) using restriction enzymes formed by PCR primers.

Various individual light and heavy chain clones were sequenced by conventional means to recognize and possible polymerases generated sequence errors (FIGS. 12 and 13). Using the Chothia canonical classification definition, three light and heavy chain CDRs were recognized (FIGS. 12-14).

A search of the NCBI IgBlast database showed that the heavy chain variable region was derived from the IgVh J558.c gonadogene, while the anti-IGF-I receptor antibody light chain variable region was derived from the mouse IgVk Cr1 gonadogene (FIG. 15).

Protein sequencing of the murine EM164 antibody was performed to confirm the sequences shown in FIGS. 12 and 13. The heavy and light chain protein bands of the purified EM164 antibody were transferred from the gel to the PVDF membrane (SDS-PAGE, reducing conditions), run from the PVDF membrane and analyzed by protein sequencing. The N-terminal sequence of the light chain was determined by Edman sequencing: DVLMTQTPLS (SEQ ID NO: 20), consistent with the N-terminal sequence of the cloned light chain gene obtained from EM164 hybridomas.

The N-terminus of the heavy chain was found to be blocked for Edman protein sequencing. Tryptic digested peptide fragments of heavy chain of 1129.5 (M + H +, monoisotopic) were fragmented via post-source reduction (PSD) and the sequence was determined to be GRPDYYGSSK (SEQ ID NO: 21). Another tryptic digested peptide fragment of the heavy chain of 2664.2 (M + H +, monoisotopic) was also fragmented via post-source reduction (PSD) and the sequence was identified as SSSTAYMQLSSLTSEDSAVYYFAR (SEQ ID NO: 22). All of these sequences perfectly match CDR3 and Structural Part 3 (FR3) of the cloned heavy chain gene.

I. Recombinant Expression of EM164 Antibody

Light and heavy chain pair sequences were cloned into a single mammalian expression vector (FIG. 16). PCR primers for human variable regions generated restriction sites that allow human signal sequences to bind while the pBluescriptII clone is in the vector, and the variable sequences express mammalian expression using EcoRI and BsiWI or HindIII and ApaI sites for light or heavy chains, respectively. Cloned into plasmid (FIG. 16). The light chain variable sequences were cloned into the structure on human IgK constant regions and the heavy chain variable sequences were cloned into human Iggamma1 constant region sequences. In the final expression plasmid, human CMV promoters promoted expression of both light and heavy chain cDNA sequences. Expression and purification of recombinant mouse EM164 antibody was carried out according to the art.

Example  2: EM164  Humanized version of the antibody

Resurfacing of EM164 antibodies to provide a suitable humanized version as a therapeutic or diagnostic agent generally proceeds according to the principles and methods disclosed in US Pat. No. 5,639,641 and the following methods.

A. Surface part prediction

Solvent accessibility of variable region sites to a group of antibodies with dissolved structures was used to predict surface site sites for murine anti-IGF-I receptor antibody (EM164) variable regions. Amino acid solvent accessibility for a set of 127 specific antibody structure files (Table 2) was calculated with the MC software package (Pedersen et al., 1994, J. Mol. Biol., 235, 959-973). The ten most similar light and heavy chain amino acid sequences from this set of 127 structures were determined by sequence sequence. Average solvent accessibility was calculated for each variable region site and positions of 30% or more of average accessibility were considered surface site sites. Locations with average accessibility between 25% and 35% were also examined to yield respective site accessibility for such structures only.

TABLE 2 term- IGF -I-receptor antibody ( EM164 )of Surface  To predict Brookhaven  127 antibody structures from the database

127 used for surface prediction Brookhaven  Structure file 2rcs 3hfl 3hfm 1aif 1a3r 1bbj 43c9 4fab 6fab 7fab 2gfb 2h1p 2hfl 1a6t 1axt 1bog 2hrp 2jel 2mcp 2pcp 1yuh 2bfv 2cgr 8fab 1ae6 1bvl 2dbl 2f19 2fb4 2fbj 1sm3 1tet 1vfa glb2 1a4j 1cly 1vge 1yec 1yed 1yee 1 nsn 1opg 1osp 1aj7 1ay1 1clz 1plg 1psk 1rmf 1sbs 1ncd 1nfd 1ngp 1acy 1afv 1cbv 1nld 1nma 1 nmb 1nqb 1mcp 1mfb 1 mim 15c8 1a5f 1axs 1mlb 1 mpa 1nbv 1ncb 1jrh 1kb5 1kel 1ap2 1b2w 1adq 1kip 1kir 1lve 1mam 1igi 1igm 1igt 1ad0 1baf 1cfv 1igy 1ikf 1jel 1jhl 1gpo 1hil 1hyx 1a0q 1bjm 1clo 1iai 1ibg 1igc 1igf 1fpt 1frg 1fvc 1aqk 1bln 1d5b 1gaf 1ggi 1ghf 1gig 1fai 1fbi 1fdl 1ad9 1bbd 1f58 1fgv 1fig 1flr 1for 1dbl 1dfb 1a3l 1bfo 1eap 1dsf 1dvf

B. molecule modelling :

Molecular models of rat EM164 were formed using the Oxford molecular software package AbM. The antibody structure was generated from the structural file for the antibody with the most similar amino acid sequence, with the sequence 2jel for the light chain and 1nqb for the heavy chain. Non-canonical CDRs were generated by searching the C-alpha structure database containing non-redundant dissolved structures. Sites placed in the CDRs within 5 ms were determined.

C. Human Antibody Selection

Surface locations of murine EM164 were compared with corresponding positions in human antibody sequences in the Kabat database (Johnson, G. and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206). Antibody database management software SR (Searle 1998) was used to extract and align antibody surface sites from natural heavy and light chain human antibody pairs. The human antibody surface portion with the most consistent surface portion was chosen to replace the murine anti-IGF-I-receptor antibody surface portion.

D. PCR Mutation

PCR mutations were performed to generate human EM164 resurfaced on murine EM164 cDNA clones (above). Primer sets were designed to form the 8 amino acid changes required for all tested versions of huEM164, and additional primers were alternatively designed to form two 5-digit changes (Table 3). The PCR reaction was carried out with the following programs: 1) 94 ° C 1 minute, 2) 94 ° C 15 seconds, 3) 55 ° C 1 minute, 4) 72 ° C 1 minute, 5) 29 cycles back to step # 2, 6 ) End to last extension step at 72 ° C. for 4 minutes. The PCR product was digested with the relevant restriction enzyme and cloned as above with the pBluescript cloning vector. Clones were sequenced to identify desirable amino acid changes.

Table 3 4 Humanization EM164  Used to generate antibodies PCR primer

primer order Sequence number : Em164hcvv CAGGTGTACACTCCCAGGTCCAACTGGTGCAGTCTGGGG CTGAAGTGGTGAAGCCTG 23 Em164hcqqgol1 CAATCAGAAGTTCCAGGGGAAGGCCACAC 24 Em164hcqqgol2 CCTTCCCCTGGAACTTCTGATTGTAGTTAGTACG 25 Em164lcv3 CAGGTGTACACTCCGATGTTGTGATGACCCAAACTCC 26 Em164lcl3 CAGGTGTACACTCCGATGTTTTGATGACCCAAACTCC 27 Em164lcp18 GACTAGATCTGCAAGAGATGGAGGCTGGATCTCCAAGAC 28 Em164lcbgl2 TTGCAGATCTAGTCAGAGCATAGTACATAGTAATG 29 Em164r45 GAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAGGCTC CTGATCTAC 30 Em164a67ol1 GTGGCAGTGGAGCAGGGACAGATTTCAC 31 Em164a67ol2 GAAATCTGTCCCTGCTCCACTGCCACTG 32

E. Variable area Surface part Seat

Pedersen et al. (J. Mol. Biol., 235, 959-973, 1994) and Roguska et al. The antibody resurfacing technique described by (Protein Eng., 9, 895-904, 1996) begins by predicting the surface portion of the murine antibody variable sequence. Surface sites are defined as amino acids having at least 30% of the total surface area accessible to water molecules. The ten most homologous antibodies were identified within the 127 antibody structure file set (FIGS. 17 and 18). Solvent accessibility for each Kabat position was averaged for these ligand sequences and the relative accessibility for each position is shown in FIG. 19. Both light and heavy chains had 26 sites with an average relative access of at least 30% (FIG. 19): thus these sites were the surface site predicted for EM164. Various sites had an average accessibility between 25% and 35%, and they were also examined averaging only antibodies with two identical sites flanking one side of the site (Tables 4 and 5). After this further analysis, the identified initial surface sets remained unchanged.

Table 4 EM164 Antibody Light chain  And Heavy chain  For variable region sequences Surface  Digit and average accessibility ( ave . acc .)

Table 5

 Sites with average accessibility between 25% and 35% were also examined by averaging a subset of antibodies with two matching sites flanking one side of the site in question. Given average accessibility, NA's mean no matching flanking sites in the 10 most homologous antibodies)

F. Molecular modeling to determine the site of CDRs within 5 ms

The molecular model was generated with the AbM software package and analyzed to determine if the EM164 surface sites are within 5 kHz CDR. In order to resurface murine EM164 antibodies, all of the surface sites outside the CDRs will have to be altered to human counterparts, but sites within 5 ′ CDRs are treated specially because they contribute to antigen specificity. Therefore, these latter positions must be identified and carefully considered throughout the humanization process. The CDR definitions used for resurfacing combine the AbM definitions for the heavy chain cdr2 and the Kabat definitions for the five remaining CDRs (FIG. 14). Table 6 shows the sites that were in CDR sites within 5 ms of either the light or heavy chain sequence of the EM164 model.

Table 6. 5Å CDR  Within EM164  Antibody Structural Surface Sites

G. Recognition of the most homologous human surface parts

Appropriate human antibody surfaces for resurfacing EM164 were recognized using SR software within the Kabat antibody sequence database, and the software provided results that searched only for specific site locations corresponding to the antibody database. In order to preserve natural pairings, surface sites of both the light and heavy chains were compared together. The most homologous human surface parts from the Kabat database were arranged in sequence homology. The top five surface parts are given in Table 7. These surfaces were then compared to the homology of those requiring minimal changes in CDRs within 5 ms. The leukemia B-cell antibody, CLL 1.69, required minimal surface site changes (total 10), and only two sites were in the CDRs within 5 ms.

The full length variable region sequence for EM164 was also arranged corresponding to the Kabat human antibody database, and CLL 1.69 was again recognized as the most similar human variable region sequence. All in all, this sequence comparison recognized the human leukemia B-cell antibody CLL 1.69 as a preferred choice as the human surface portion for EM164.

Table 7- Kabat From database  Top 5 Human Sequences Extracted

(Arrays were generated by SR (Pedersen 1993). EM164 surface sites with CDRs within 5 μs are underlined.)

H. Humanization EM164 Gene composition

Ten surface site changes for EM164 were formed using the PCR mutation technique described above (Table 7). Since 8 of the surface sites in CLL 1.69 were not CDRs within 5 ms, these sites were altered in all humanized EM164 versions from rat to human (Tables 8 and 9). The two light chain surface sites in the CDRs within 5 μs (Kabat positions 3 and 45) were changed to human or maintained in rats. Together, these choices result in four humanized versions of the EM164 formed (FIGS. 22 and 23).

Of the four humanized versions, version 1.0 has all ten human surface sites. The most fixed version of the marginal change in the CDRs is version 1.1, which retained both of the mouse surface sites that were within the CDRs within 5 ms. All four humanized EM164 antibody genes were cloned into antibody expression plasmids for use in transient and immobilized transmission (FIG. 16).

Table 8-Humanization EM164  Site Changes in Versions 1.0-1.3 of Antibodies

I. total length IGF -I receptor and cleaved IGF Humanization in Binding to the I-I Receptor Alpha Chain EM164 Antibody Versions of Mice EM164 Comparison of affinity with antibodies

The affinity of humanized EM164 antibody versions 1.0-1.3 was determined by binding competition experiments using biotinylated full-length human IGF-I receptor or myc-antigenic determinant labeled cleaved IGF-I receptor alphachain. Contrasted with the affinity of. Humanized EM164 antibody specimens were obtained by transient transmission of appropriate expression vectors in human embryonic kidney 293T cells, and antibody concentrations were determined using a humanized antibody standard purified by ELISA. For ELISA binding competition measurements, mixtures of various concentrations of humanized antibody specimens and murine EM164 antibodies were indirectly captured biotinylated full-length IGF-I receptor or myc-antigenic determinant labeled cleaved IGF-I receptor alphachain Were incubated. After equilibrium, bound humanized antibodies were found using goat-anti-human-fab'2-antibody-horseradish peroxidase conjugates. A plot of ([bound murine antibody] / [bound humanized antibody]) versus ([murine antibody] / [humanized antibody]) yields a theoretically straight line = (K _{d humanized antibody} / K _{d Murine antibodies} ), humanized and the relative affinity of murine antibodies.

Exemplary competitive experiments are shown in FIG. 11. Immulon-2HB ELISA plates were coated with 100 ml of 5 mg / ml streptavidin per well in carbonate buffer at ambient temperature for 7 hours. Streptavidin-coated wells were blocked for 1 hour with 200 ml of blocking buffer (10 mg / ml BSA in TBS-T buffer), washed with TBS-T buffer, and biotinylated IGF- at 4 ° C. overnight. Incubated with I receptor (5 ng per well).

Wells containing indirectly captured biotinylated IGF-I receptors are then washed and humanized EM164 antibody (15.5 ng) and murine antibodies (0 ng, or 16.35 ng, or 32.7 ng, or 65.4 ng, or 163.5 ng) Incubated at ambient temperature for 2 hours in 100 mL blocking buffer with mixtures of then incubated overnight at 4 ° C. Wells were then washed with TBS-T buffer and incubated with goat-anti-human-fab'2-antibody-horseradish peroxidase conjugate for 1 hour (100 ml; 1 mg / ml in blocking buffer), washed and Detection with ABTS / H ₂ O ₂ was followed at 405 nm.

The composition of ([Ru] antibody / [bound humanized antibody]) versus ([Ru] antibody / [humanized antibody]) produced a straight line with a slope of 0.52 (= K _{d humanized antibody} / K _{d murine antibody} ) (r ² = 0.996). Therefore, humanized antibody version 1.0 binds more tightly to the IGF-I receptor as compared to murine EM164 antibody. Similar values in the range of 0.5 to 0.8 for competition in binding to the full-length IGF-I receptor or truncated IGF-I receptor alpha chains with murine EM164 antibodies of versions 1.0, 1.2, 1.3 of humanized EM164 antibodies Obtained, indicating that all humanized versions of EM164 antibodies have similar affinities and are better than for parental mouse EM164 antibodies. The Shimeric version of the EM164 antibody with the 92F → C mutation in the heavy chain showed a slope of about 3 in a similar binding competition with the murine EM164 antibody, indicating that the 92F → C mutation of the EM164 antibody was more IGF-I than the murine EM164 antibody. 3 times lower affinity for the body. Humanized EM164 v1.0 antibody showed similar inhibition of IGF-I-promoted growth and survival of MCF-7 cells as did the rat EM164 antibody (FIG. 24). Inhibition of serum-promoting growth and survival of MCF-7 cells by humanized EM164 v1.0 antibody was similar to that by rat de164 antibody.

Table 9

snippet Light chain Heavy chain FR1 1-23 (with an occasional residue at 0, and a deletion at 10 in V _l chains) 1-30 (with an occasional residue at 0) CDR1 24-34 (with possible insertions numbered as 27A, B, C, D, E, F) 31-35 (with possible insertions numbered as 35A, B) FR2 35-49 36-49 CDR2 50-56 50-65 (with possible insertions numbered as 52A, B, C) FR3 57-88 66-94 (with possible insertions numbered as 82A, B, C) CDR3 89-97 (with possible insertions numbered as 95A, B, C, D, E, F) 95-102 (with possible insertions numbered as 100A, B, C, D, E, F, G, H, I, J, K) FR4 98-107 (with a possible insertion numbered as 106A) 103-113

(Kabat systems are used for the light and heavy chain variable region polypeptides of different versions of EM164 antibody. Amino acid sites are united into structural (FR) and complementarity determining regions (CDR) depending on their position in the polypeptide chain.

Kabat et al. Sequences of Proteins of Immunological Interest , Fifth Edition, 1991, NIH Publication No. Collected as 91-3242)

J. Improved anti- starting with the mouse and human and antibody sequences described herein IGF Processes for Providing -I Receptor Antibodies:

The amino acid and nucleic acid sequences of the anti-IGF-I receptor antibody EM164 and its humanized variants have been used to express other antibodies with improved properties and also within the scope of the present invention. Improved properties include increased affinity for the IGF-I receptor. Various studies have shown the effect of having one or more amino acid changes at various positions within an antibody sequence based on knowledge of primary antibody sequences on properties such as binding and expression levels (Yang, WP et al., 1995). , J. Mol . Biol . , 254, 392-403; Rader, C. et al., 1998, Proc . Natl . Acad . Sci . USA , 95, 8910-8915; Vaughan, TJ et al., 1998, Nature Biotechnology , 16, 535-539). In these studies, the variants of the primary antibody are heavy chains in CDR1, CDR2, CDR3, or structural regions using methods such as oligonucleotide-mediated relocation mutations, cassette mutations, error-prone PCR, DNA shuffling, or E. coli mutant strains. and it was produced by changing the sequence of the light chain gene (Vaughan, TJ et al, 1998 , Nature Biotechnology, 16, 535-539;... Adey, NB et al, 1996, Chapter 16, pp 277-291, in "Phage Display of Peptides and Proteins " , Eds. Kay, BK et al., Academic Press). These primary antibody sequence alteration methods resulted in improved affinity of secondary antibodies through standard detection techniques (Gram, H. et al., 1992, Proc. ... Natl Acad Sci USA, 89, 3576-3580;.. Boder, ET et al, 2000, Proc Natl Acad Sci USA, 97, 10701-10705;.... Davies, J. and Riechmann, L., 1996, Immunotechnolgy , 2, 169-179; Thompson, J. et al., 1996, J. Mol . Biol . , 256, 77-88; Short, MK et al., 2002, J. Biol . Chem . , 277 , 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem . , 276, 27622-27628).

By a similar design method of changing one or more amino acid sites, the antibody sequences described herein may be used, such as antibodies having an appropriate group, such as amino- or thiol-free groups at the binding site for covalent modification, for example at the therapeutic binding site. It can be used to express anti-IGF-I receptor antibodies with improved function.

K. Alternative Expression Systems for Rat, Shimeric and Other Anti- IGF- I Receptor Antibodies

Murine anti IGF-I receptor antibodies were also expressed from mammalian expression plasmids similar to those used to express humanized antibodies (above). Expression plasmids are known to have murine constant regions comprising light chain κ and heavy chain γ-1 sequences (McLean et al., 2000, Mol Immunol ., 37, 837-845). These plasmids were designed by single restriction digestion and cloning to accept antibody variable regions such as, for example, murine anti-IGF-I receptor antibodies. Additional PCR of anti-IGF-I receptor antibodies was generally required to form restrictions that would correspond with those in the expression plasmid.

An alternative approach to fully express the murine anti-IGF-I receptor antibody was to replace the human constant region in the Shimeric anti-IGF-I receptor antibody expression plasmid. Shimeric expression plasmids (FIG. 16) were formed using cassettes for both variable and light and heavy chain constant regions. Separate restriction digests were used for cloning within any constant region sequence, as antibody variable region sequences were cloned into expression plasmids by restriction digests. κ light chain and γ-1 heavy chain cDNAs were cloned from murine hybridoma RNA as described herein for example for cloning the anti-IGF-I antibody variable region. Similarly suitable primers were devised from sequences available in the Kabat database (see Table 10). For example, RT-PCR was used for cloning of constant region sequences and for forming restriction sites necessary for cloning fragments into Shimeric anti-IGF-I receptor antibody expression plasmids. The plasmid was then used to fully express anti-IGF-I receptor antibodies in a rat standard mammalian expression system, such as the CHO cell line.

Table 10-Rat γ-1 constant region and rat κ constant region, respectively Cloning  Designed for primer .

Rat constant region primer Primer Name Primer sequence SEQ ID NO: MuIgG1 C3endX TTTTGAGCTCTTATTTACCAGGAGAGTGGGAGA GGCTCTT 45 MuIgG1 C5endH TTTTAAGCTTGCCAAAACGACACCCCCATCTGTCTAT 46 MuIgKap C3endB TTTTGGATCCTAACACTCATTCCTGTTGAAGC 47 MuIgKap C5endE TTTTGAATTCGGGCTGATGCTGCACCAACTG 48

Primers were designed from sequences obtainable from Kabat database (Johnson, G and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206).

Statement of Deposit

Hybridomas making murine EM164 antibodies were deposited with the American Type Culture Collection, PO Box 1549, Manassas, VA 20108 on June 14, 2002, in accordance with the Budapest Convention on Microbial Deposits and the accredited ATCC accession number PTA-4457.

Certain patents and printed publications are referred to in this disclosure (indicating that each of them is incorporated by reference in its entirety).

Although the invention has been described in detail and with reference to specific embodiments, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

 <110> ImmunoGen, Inc.   <120> ANTI-IGF-I RECEPTOR ANTIBODY <130> F190322 <140> 10 / 729,441 <141> 2003-12-08 <150> 10 / 170,390 <151> 2002-06-14 <160> 98 <170> PatentIn version 3.3 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain complementarity determining region <400> 1 Ser Tyr Trp Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain complementarity determining region <400> 2 Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Arg      <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain complementarity determining region <400> 3 Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp Val 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain complementarity determining region <400> 4 Arg Ser Ser Gln Ser Ile Val His Ser Asn Val Asn Thr Tyr Leu Glu 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain complementarity determining region <400> 5 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain complementarity determining region <400> 6 Phe Gln Gly Ser His Val Pro Pro Thr 1 5 <210> 7 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> antibody heavy chain <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> antibody light chain <400> 8 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 9 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 10 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 10 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 11 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 11 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 12 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized light chain variable region <400> 12 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 13 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> humanized heavy chain variable region <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe     50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 14 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> degenerate 3 'light chain PCR primer-HindKL <400> 14 tatagagctc aagcttggat ggtgggaaga tggatacagt tggtgc 46 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> degenerate 3 'heavy chain PCR primer- Bgl2IgG1 <400> 15 ggaagatcta tagacagatg ggggtgtcgt tttggc 36 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> poly C 5 'PCR primer-EcoPolyC <400> 16 tatatctaga attccccccc cccccccccc 30 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> degenerate 5 'light chain PCR primer-Sac1MK <400> 17 gggagctcga yattgtgmts acmcarwctm ca 32 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> degenerate 5 'heavy chain PCR primer-EcoR1MH1 <220> <221> misc_feature (222) (18) .. (18) <223> "n" may be any nucleic acid <400> 18 cttccggaat tcsargtnma gctgsagsag tc 32 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> degenerate 5 'heavy chain PCR primer-EcoR1MH2 <220> <221> misc_feature (222) (18) .. (18) <223> "n" may be any nucleotide <400> 19 cttccggaat tcsargtnma gctgsagsag tcwgg 35 <210> 20 <211> 10 <212> PRT <213> Mus musculus <400> 20 Asp Val Leu Met Thr Gln Thr Pro Leu Ser 1 5 10 <210> 21 <211> 10 <212> PRT <213> Mus musculus <400> 21 Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys 1 5 10 <210> 22 <211> 24 <212> PRT <213> Mus musculus <400> 22 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 1 5 10 15 Ser Ala Val Tyr Tyr Phe Ala Arg             20 <210> 23 <211> 57 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 23 caggtgtaca ctcccaggtc caactggtgc agtctggggc tgaagtggtg aagcctg 57 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 24 caatcagaag ttccagggga aggccacac 29 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 25 ccttcccctg gaacttctga ttgtagttag tacg 34 <210> 26 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 26 caggtgtaca ctccgatgtt gtgatgaccc aaactcc 37 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 27 caggtgtaca ctccgatgtt ttgatgaccc aaactcc 37 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 28 gactagatct gcaagagatg gaggctggat ctccaagac 39 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 29 ttgcagatct agtcagagca tagtacatag taatg 35 <210> 30 <211> 48 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 30 gaatggtacc tgcagaaacc aggccagtct ccaaggctcc tgatctac 48 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 31 gtggcagtgg agcagggaca gatttcac 28 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 32 gaaatctgtc cctgctccac tgccactg 28 <210> 33 <211> 19 <212> PRT <213> Homo sapiens <400> 33 Asp Leu Thr Leu Leu Gln Pro Gly Gln Lys Gly Asp Ser Arg Glu Lys 1 5 10 15 Lys Arg Ala              <210> 34 <211> 18 <212> PRT <213> Homo sapiens <400> 34 Asp Val Thr Leu Leu Pro Pro Gly Gln Arg Gly Asp Ala Arg Glu Lys 1 5 10 15 Lys arg          <210> 35 <211> 19 <212> PRT <213> Homo sapiens <400> 35 Asp Gln Ser Leu Ile Pro Pro Gly Gln Lys Gly Asp Ser Arg Asp Lys 1 5 10 15 Lys Arg Ala              <210> 36 <211> 18 <212> PRT <213> Homo sapiens <400> 36 Asp Met Ser Ser Val Arg Pro Gly Gln Lys Gly Ser Ser Ser Asp Lys 1 5 10 15 Lys arg          <210> 37 <211> 18 <212> PRT <213> Homo sapiens <400> 37 Glu Val Ser Gly Pro Arg Pro Gly Gln Arg Gly Asp Ser Arg Glu Lys 1 5 10 15 Lys arg          <210> 38 <211> 18 <212> PRT <213> Homo sapiens <400> 38 Glu Val Ser Gly Pro Arg Pro Gly Gln Arg Gly Asp Ser Arg Glu Lys 1 5 10 15 Lys arg          <210> 39 <211> 25 <212> PRT <213> Homo sapiens <400> 39 Gln Gln Gln Ala Leu Lys Pro Gly Lys Lys Thr Pro Gly Gln Glu Lys 1 5 10 15 Lys Arg Lys Ser Ser Ser Glu Ala Ser             20 25 <210> 40 <211> 25 <212> PRT <213> Homo sapiens <400> 40 Gln Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys 1 5 10 15 Gln Gly Lys Ser Ser Ser Glu Gln Ser             20 25 <210> 41 <211> 25 <212> PRT <213> Homo sapiens <400> 41 Gln Gln Gln Pro Leu Lys Pro Gly Lys Lys Thr Pro Gly Lys Asp Asp 1 5 10 15 Lys Gly Thr Ser Asn Asn Glu Gln Ser             20 25 <210> 42 <211> 25 <212> PRT <213> Homo sapiens <400> 42 Gln Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys 1 5 10 15 Lys Gly Lys Ser Ser Ser Glu Gln Ser             20 25 <210> 43 <211> 24 <212> PRT <213> Homo sapiens <400> 43 Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys Gln 1 5 10 15 Gly Lys Ser Ser Ser Glu Gln Ser             20 <210> 44 <211> 24 <212> PRT <213> Homo sapiens <400> 44 Gln Val Ala Val Lys Pro Gly Lys Lys Thr Pro Gly Gln Gln Lys Gln 1 5 10 15 Gly Glu Ser Ser Ser Glu Gln Ser             20 <210> 45 <211> 40 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 45 ttttgagctc ttatttacca ggagagtggg agaggctctt 40 <210> 46 <211> 37 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 46 ttttaagctt gccaaaacga cacccccatc tgtctat 37 <210> 47 <211> 32 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 47 ttttggatcc taacactcat tcctgttgaa gc 32 <210> 48 <211> 31 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 48 ttttgaattc gggctgatgc tgcaccaact g 31 <210> 49 <211> 396 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (396) <400> 49 atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct gct 48 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 tcc agt agt gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc 96 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val             20 25 30 agt ctt gga gat caa gcc tcc atc tct tgc aga tct agt cag agc att 144 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile         35 40 45 gta cat agt aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca 192 Val His Ser Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro     50 55 60 ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct 240 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 ggg gtc cca gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca 288 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr                 85 90 95 ctc agg atc agc aga gtg gag gct gag gat ctg gga att tat tac tgc 336 Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys             100 105 110 ttt caa ggt tca cat gtt cct ccg acg ttc ggt gga ggc acc aag ctg 384 Phe Gln Gly Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu         115 120 125 gaa atc aaa cgg 396 Glu Ile Lys Arg     130 <210> 50 <211> 132 <212> PRT <213> Mus musculus <400> 50 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val             20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile         35 40 45 Val His Ser Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro     50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr                 85 90 95 Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys             100 105 110 Phe Gln Gly Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu         115 120 125 Glu Ile Lys Arg     130 <210> 51 <211> 429 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (429) <400> 51 atg gga tgg agc tat atc atc ctc ttt ttg gta gca aca gct aca gaa 48 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Glu 1 5 10 15 gtc cac tcc cag gtc caa ctg cag cag tct ggg gct gaa ctg gtg aag 96 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys             20 25 30 cct ggg gct tca gtg aag ctg tcc tgt aag gct tct ggc tac acc ttc 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe         35 40 45 acc agc tac tgg atg cac tgg gtg aag cag agg cct gga caa ggc ctt 192 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu     50 55 60 gag tgg att gga gag att aat cct agc aac ggt cgt act aac tac aat 240 Glu Trp Ile Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 gag aag ttc aag agg aag gcc aca ctg act gta gac aaa tcc tcc agc 288 Glu Lys Phe Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser                 85 90 95 aca gcc tac atg caa ctc agc agc ctg aca tct gag gac tct gcg gtc 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 tat tac ttt gca aga gga aga cca gat tac tac ggt agt agc aag tgg 384 Tyr Tyr Phe Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp         115 120 125 tac ttc gat gtc tgg ggc gca ggg acc acg gtc acc gtc tcc tca 429 Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser     130 135 140 <210> 52 <211> 143 <212> PRT <213> Mus musculus <400> 52 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Glu 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys             20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe         35 40 45 Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu     50 55 60 Glu Trp Ile Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Phe Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser                 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val             100 105 110 Tyr Tyr Phe Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp         115 120 125 Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser     130 135 140 <210> 53 <211> 10 <212> PRT <213> Mus musculus <400> 53 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 54 <211> 10 <212> PRT <213> Mus musculus <400> 54 Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn 1 5 10 <210> 55 <211> 15 <212> PRT <213> Mus musculus <400> 55 Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp Val 1 5 10 15 <210> 56 <211> 100 <212> PRT <213> Mus musculus <400> 56 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro             100 <210> 57 <211> 98 <212> PRT <213> Mus musculus <400> 57 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala arg          <210> 58 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 58 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 59 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 59 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Ser Ile Ser Ser Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Gln Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 60 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 60 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Thr His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 61 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 61 Asp Ile Glu Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 62 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 62 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Phe Ser Gln Ser Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Ser Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 63 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 63 Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser             20 25 30 Asn Gly Asp Thr Tyr Leu Asp Trp Phe Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 64 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 64 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Asn Gln Thr Ile Leu Leu Ser             20 25 30 Asp Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 65 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 65 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val His Ser             20 25 30 Ser Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Ile Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 66 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 66 Asp Val Leu Met Thr Gln Ile Pro Val Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Val His Asn             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 67 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 67 Asp Val Leu Met Thr Gln Thr Pro Val Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Thr Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala                 85 90 95 Ser His Ala Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 68 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 68 Asp Val Leu Met Thr Gln Ile Pro Val Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile Val His Asn             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 69 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <220> <221> MISC_FEATURE (222) (28) .. (28) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (101) .. (101) <223> "X" may be any amino acid <400> 69 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Xaa Ile Val His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Xaa Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 70 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 70 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 71 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 71 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile         35 40 45 Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 72 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 72 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile         35 40 45 Gly Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Tyr Asp Tyr Tyr Gly Ser Ser Tyr Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Leu Thr Val Ser Ser         115 120 <210> 73 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 73 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Gly Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Pro Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Ser Leu Tyr Tyr Tyr Gly Thr Ser Tyr Gly Val Leu Asp Tyr Trp             100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 74 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 74 Gln Val Gln Leu Gln Gln Pro Gly Ser Val Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser             20 25 30 Trp Ile His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile His Pro Asn Ser Gly Asn Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Val Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Trp Arg Tyr Gly Ser Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Leu Thr Val Ser Ser         115 120 <210> 75 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 75 Gln Val Gln Phe Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Leu Met His Trp Ile Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile         35 40 45 Gly Arg Ile Asp Pro Asn Asn Val Val Thr Lys Phe Asn Glu Lys Phe     50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Tyr Ala Tyr Cys Arg Pro Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Thr Val Thr Val Ser Ser         115 <210> 76 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 76 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr             20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Glu Gly Leu Glu Trp Ile         35 40 45 Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys                 85 90 95 Ala Arg Gly Gly Lys Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Thr Val Ser Ser         115 <210> 77 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 77 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Phe             20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Gly Thr His Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly His Ser Tyr Tyr Phe Tyr Asp Gly Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 78 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 78 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr             20 25 30 Tyr Ile Asn Trp Met Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Trp Ile Asp Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys                 85 90 95 Ala Arg Glu Lys Thr Thr Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Ser Val Thr Val Ser Ala         115 120 <210> 79 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 79 Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Phe             20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Gly Thr His Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly His Ser Tyr Tyr Phe Tyr Asp Gly Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 80 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <400> 80 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr Trp             20 25 30 Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly         35 40 45 Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr His Glu Arg Phe Lys     50 55 60 Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Gly Val Tyr Tyr Cys Leu                 85 90 95 His Gly Asn Tyr Asp Phe Asp Gly Trp Gly Gln Gly Thr Thr Leu Thr             100 105 110 Val Ser Ser         115 <210> 81 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> synthetic antibody structure <220> <221> MISC_FEATURE (222) (20) .. (20) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (34) .. (34) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (43) .. (43) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (50) .. (50) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (52) .. (52) <223> "X" may be any amino acid <220> <221> MISC_FEATURE <222> (54) .. (54) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (57) .. (57) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (59) .. (59) <223> "X" may be any amino acid <220> <221> MISC_FEATURE (222) (99) .. (99) <223> "X" may be any amino acid <220> <221> MISC_FEATURE <222> (100) .. (100) <223> "X" may be any amino acid <220> <221> MISC_FEATURE <222> (103) .. (108) <223> "X" may be any amino acid <220> <221> MISC_FEATURE 116 (116) .. (116) <223> "X" may be any amino acid <400> 81 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Xaa His Trp Val Lys Gln Arg Pro Gly Xaa Gly Leu Glu Trp Ile         35 40 45 Gly Xaa Ile Xaa Pro Xaa Ser Gly Xaa Thr Xaa Tyr Asn Glu Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Val Tyr Cys                 85 90 95 Ala Arg Xaa Xaa Tyr Tyr Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Xaa Val Thr Val Ser Ser         115 120 <210> 82 <211> 113 <212> PRT <213> Mus musculus <400> 82 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 83 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 83 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 84 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 84 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 85 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 85 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 86 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 86 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 87 <211> 123 <212> PRT <213> Mus musculus <400> 87 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe     50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser         115 120 <210> 88 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> humanized EM164 antibody <400> 88 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe     50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser         115 120 <210> 89 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> variable region of humanized EM164 antibody-light chain <220> <221> CDS (222) (1) .. (339) <400> 89 gat gtt gtg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cca gcc tcc atc tct tgc aga tct agt cag agc ata gta cat agt 96 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 cca agg ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 gac agg ttc agt ggc agt gga gca ggg aca gat ttc aca ctc agg atc 240 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga att tat tac tgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 tca cat gtt cct ccg acg ttc ggt gga ggc acc aaa ctg gaa atc aaa 336 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 cgt 339 Arg                                                                          <210> 90 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 90 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 91 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> variable region of humanized EM164 antibody-heavy chain <220> <221> CDS (222) (1) .. (369) <400> 91 cag gtc caa ctg gtg cag tct ggg gct gaa gtg gtg aag cct ggg gct 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 tca gtg aag ctg tcc tgt aag gct tct ggc tac acc ttc acc agc tac 96 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 tgg atg cac tgg gtg aag cag agg cct gga caa ggc ctt gag tgg att 144 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 gga gag att aat cct agc aac ggt cgt act aac tac aat cag aag ttc 192 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe     50 55 60 cag ggg aag gcc aca ctg act gta gac aaa tcc tcc agc aca gcc tac 240 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg caa ctc agc agc ctg aca tct gag gac tct gcg gtc tat tac ttt 288 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 gca aga gga aga cca gat tac tac ggt agt agc aag tgg tac ttc gat 336 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 369 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser         115 120 <210> 92 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 92 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Gln Lys Phe     50 55 60 Gln Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Phe                 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Gly Ser Ser Lys Trp Tyr Phe Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser         115 120 <210> 93 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of humanized EM164 v1.1 antibody <220> <221> CDS (222) (1) .. (339) <400> 93 gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cca gcc tcc atc tct tgc aga tct agt cag agc ata gta cat agt 96 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 gac agg ttc agt ggc agt gga gca ggg aca gat ttc aca ctc agg atc 240 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga att tat tac tgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 tca cat gtt cct ccg acg ttc ggt gga ggc acc aaa ctg gaa atc aaa 336 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 cgt 339 Arg                                                                          <210> 94 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 94 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 95 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of humanized EM164 v1.2 antibody <220> <221> CDS (222) (1) .. (339) <400> 95 gat gtt ttg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cca gcc tcc atc tct tgc aga tct agt cag agc ata gta cat agt 96 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 cca agg ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 gac agg ttc agt ggc agt gga gca ggg aca gat ttc aca ctc agg atc 240 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga att tat tac tgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 tca cat gtt cct ccg acg ttc ggt gga ggc acc aaa ctg gaa atc aaa 336 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 cgt 339 Arg                                                                          <210> 96 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 96 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg      <210> 97 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> light chain variable region of humanized EM164 v1.3 antibody <220> <221> CDS (222) (1) .. (339) <400> 97 gat gtt gtg atg acc caa act cca ctc tcc ctg cct gtc agt ctt gga 48 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat cca gcc tcc atc tct tgc aga tct agt cag agc ata gta cat agt 96 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 aat gta aac acc tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 gac agg ttc agt ggc agt gga gca ggg aca gat ttc aca ctc agg atc 240 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 agc aga gtg gag gct gag gat ctg gga att tat tac tgc ttt caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 tca cat gtt cct ccg acg ttc ggt gga ggc acc aaa ctg gaa atc aaa 336 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 cgt 339 Arg                                                                          <210> 98 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 98 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser             20 25 30 Asn Val Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg     

Claims (31)

(a) a primary therapeutic agent, (i) an antibody or epitope-binding fragment thereof having the same amino acid sequence as mouse antibody EM164 prepared by mouse hybridoma EM164 (ATCC accession number PTA-4457), (ii) a resurfaced antibody or epitope-binding fragment thereof having the same binding specificity as murine antibody EM164, (iii) a human antibody or humanized antibody, or epitope-binding fragment thereof, having the same binding specificity as murine antibody EM164, (iv) a functional equivalent of an antibody or epitope-binding fragment thereof having the same binding specificity as murine antibody EM164, (v) a variant or epitope-binding fragment of murine antibody EM164 having at least one nucleotide mutation, deletion or insertion as compared to murine antibody EM164, and having the same binding specificity as murine antibody EM164, and (vi) is selected from the group consisting of murine antibody EM164 or epitope-binding fragment thereof prepared by mouse hybridoma EM164 (ATCC accession number PTA-4457), and is an antibody or epitope-binding fragment thereof, The antibody or fragment specifically binds to an insulin-like growth factor-I receptor, Primary treatment and (b) a composition comprising a secondary therapeutic agent. The method of claim 1, wherein the secondary therapeutic agent is docetaxel (docetaxel), paclitaxel (paclitaxel), doxorubicin (exorubicin), epirubicin (epirubicin), cyclophosphamide, trastuzumab (herceptin) )), Capecitabine, tamoxifen, toremfene, letrozole, anastrozole, fulvestrant, exemestane, high Goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin), 5-fluoro Leuuracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estramustine , Mitoxantrone, abarelix (a barelix, Zoledronate, Streptozocin, Rituximab (Rituxan), Idarubicin, Busulfan, Chlorambucil, Fludarabine, imatinib, cytarabine, ibritumomab (Zevalin), tositumomab (Bexxar), interferon α- 2b, melphalam, bortezomib (Velcade), altretamine, asparaginase, gefitinib (Iressa), eroni A composition selected from the group consisting of erlonitib (Tarceva), anti-EGF receptor antibody (Cetuximab, Abx-EGF), and epothilone. The composition of claim 1, wherein said secondary therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, and camptothecin. The method of claim 1, wherein the primary therapeutic agent is administered to the patient at a dose of about 1 mg / m ³ to about 2000 mg / m ³ , The second therapeutic agent is administered to a patient at a dose of about 10 mg / m ³ to about 2000 mg / m ³ . The method of claim 1, wherein the primary therapeutic agent is administered to the patient at a dose of about 10 mg / m ³ to about 1000 mg / m ³ , The second therapeutic agent is administered to a patient at a dose of about 50 mg / m ³ to about 1000 mg / m ³ . A pharmaceutical composition comprising the composition according to claim 1, and a pharmaceutically acceptable carrier or diluent . A pharmaceutical composition comprising the composition of claim 1, And pharmaceutically acceptable carriers or diluents . (a) a primary therapeutic agent that is an antibody or epitope-binding antibody fragment comprising at least one complementarity determining site having an amino acid sequence selected from the group consisting of: SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3), RSSQSIVHSNVNTYLE (SEQ ID NO: 4), KVSNRFS (SEQ ID NO: 5), and FQGSHVPPT (SEQ ID NO: 6), and (b) a composition comprising a secondary therapeutic agent. (a) an antibody or epitope-binding antibody fragment comprising at least one heavy chain variable region and at least one light chain variable region, Wherein the heavy chain variable region comprises three sequence complementarity determining sites each having an amino acid sequence represented by SEQ ID NOs: 1-3: SYWMH (SEQ ID NO: 1), EINPSNGRTNYNEKFKR (SEQ ID NO: 2), GRPDYYGSSKWYFDV (SEQ ID NO: 3); A primary therapeutic agent wherein said light chain variable region comprises three sequence complementarity determining sites each having an amino acid sequence represented by SEQ ID NOs: 4-6: RSSQSIVHSNVNTYLE (SEQ ID NO: 4), KVSNRFS (SEQ ID NO: 5), FQGSHVPPT (SEQ ID NO: 6), and (b) a composition comprising a secondary therapeutic agent. (a) a primary therapeutic agent wherein the antibody or fragment thereof comprises an antibody or epitope-binding fragment thereof comprising a heavy chain variable region having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 7 QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEI NPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSS KWYFDVWGAGTTVTVSS (SEQ ID NO: 7), and (b) a composition comprising a secondary therapeutic agent. 10. The composition of claim 9, wherein the heavy chain variable region has at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 7. The composition of claim 9, wherein the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 7. (a) a primary therapeutic agent wherein the antibody and fragment thereof comprise an antibody or epitope-binding fragment thereof comprising a light chain variable region having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 8: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIKR (SEQ ID NO: 8), and (b) a composition comprising a secondary therapeutic agent. 13. The composition of claim 12, wherein the light chain variable region has at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 8. The composition of claim 12, wherein the light chain variable region has an amino acid sequence represented by SEQ ID NO: 8. (a) a primary therapeutic agent wherein the antibody or fragment thereof comprises an antibody or epitope-binding fragment thereof comprising a light chain variable region having a sequence selected from the group consisting of: DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK R (SEQ ID NO: 9); DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK R (SEQ ID NO: 10); DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK R (SEQ ID NO: 11); DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIY KVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK R (SEQ ID NO: 12), and (b) a composition comprising a secondary therapeutic agent. (a) a primary therapeutic agent wherein the antibody or fragment thereof comprises a heavy chain variable region having a sequence represented by SEQ ID NO: 13 or an epitope-binding fragment thereof: QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEI NPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSS KWYFDVWGQGTTVTVSS (SEQ ID NO: 13), and (b) a composition comprising a secondary therapeutic agent. The method according to any one of claims 7 to 16, wherein the secondary therapeutic agent is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab ( Herstutin (trastuzumab (Herceptin)), capecitabine, tamoxifen, toremfene, letrozole, anastrozole, fulvestrant, fulvestrant Exemestane, goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (abastin) (bevacizumab (bevacizumab) Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine , Estramustine, mitoxant (mitoxantrone), abarelix, azoledronate, streptozocin, rituximab (rituxanb), rituxan, idarubicin, busulfan , Chlorambucil, fludarabine, imatinib, imatinib, cytarabine, ibritumab (Zevalin), tositumomab (vexar), tositumomab (Bexxar), interferon α-2b, melphalam, bortezomib (Velcade), altretamine, asparaginase, zefitinib (iresa) ( gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptor antibodies (Cetuximab, Abx-EGF), and epothilones. 17. The composition of any one of claims 7-16, wherein said secondary therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, and camptothecin. A method of inhibiting cancer cell growth comprising contacting a cell with the composition of claim 1. A method for treating a patient with cancer comprising administering an appropriate amount of the composition of claim 1 to the patient. A method for treating a patient with cancer comprising administering an appropriate amount of the pharmaceutical composition of claim 6 to the patient. The cancer according to any one of claims 19 to 21, wherein the cancer is breast cancer, colon cancer, ovarian cancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial sarcoma, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma, and rhabdomyolysis. A method selected from the group consisting of sarcomas. (a) an antibody having the same amino acid sequence as mouse antibody EM164 prepared from mouse hybridoma EM164 (ATCC accession number PTA-4457), or an epitope binding fragment thereof; A primary therapeutic agent that said antibody or fragment specifically binds to an insulin-like growth factor-I receptor, (b) secondary therapies, and (c) Kits containing instructions for use. (a) an antibody having the same amino acid sequence as mouse antibody EM164 prepared from mouse hybridoma EM164 (ATCC accession number PTA-4457), or an epitope binding fragment thereof; A primary therapeutic agent that said antibody or fragment specifically binds to an insulin-like growth factor-I receptor, and (b) inhibiting cancer cell growth comprising contacting the secondary therapeutic agent with the cell. (a) an antibody having the same amino acid sequence as mouse antibody EM164 prepared from mouse hybridoma EM164 (ATCC accession number PTA-4457), or an epitope binding fragment thereof; A primary therapeutic agent that said antibody or fragment specifically binds to an insulin-like growth factor-I receptor, and (b) A method for treating a patient with cancer comprising administering to the patient an appropriate amount of a secondary therapeutic agent. 25. The method of claim 24, wherein said cells inhibit cancer cell growth in contact with said primary and secondary therapeutic agents. he method of claim 24, wherein said cell is contacted with said first therapeutic agent and said second therapeutic agent sequentially and in either order . 25. The method of claim 24, wherein said cells inhibit cancer cell growth in contact with said primary and said second therapeutic agent sequentially and in any order. The method of claim 25, wherein the primary and second therapeutic agents are administered at the same time. 29.The method of claim 25, wherein said first therapeutic agent and said second therapeutic agent are administered sequentially and in either order . The method of claim 25, wherein the first and second therapies are administered sequentially and in any order. The method of claim 24 or 25, wherein the secondary therapeutic agent is docetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide, trastuzumab (herceptin) ( trastuzumab (Herceptin), capecitabine, tamoxifen, toremfene, letrozole, anastrozole, fulvestrant, exemestane ), Goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide, topotecan, gemcitabine, vinorelbine, estoramustine (estramustine), mitoxantrone, Abarelix, Zoledronate, Streptozocin, Rituximab (rituximab) (Rituxan), Idarubicin, Busulfan, Chlorambucil (chlorambucil), fludarabine, imatinib, cytarabine, ibritumomab (Zevalin), tositumomab (Bexxar) , Interferon α-2b, melphalam, bortezomib (Velcade), altretamine, asparaginase, gefitinib (Iressa) ), Treating patients with cancer selected from the group consisting of erlonitib (Tarceva), anti-EGF receptor antibodies (Cetuximab, Abx-EGF), and epothilones Way. The method of claim 24 or 25, wherein the secondary therapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, paclitaxel, docetaxel, gemcitabine, and camptothecin.

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