CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a Continuation Application of U.S. application Ser. No. 10/170,390, filed Jun. 14, 2002; the disclosure of which is incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to antibodies that bind to human insulin-like growth factor-I receptor (IGF-I receptor). More particularly, the invention relates to anti-IGF-I receptor antibodies that inhibit the cellular functions of the IGF-I receptor. Still more particularly, the invention relates to anti-IGF-I receptor antibodies that 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 invention also relates to fragments of said antibodies, humanized and resurfaced versions of said antibodies, conjugates of said antibodies, antibody derivatives, and the uses of same in diagnostic, research and therapeutic applications. The invention further relates to improved antibodies or fragments thereof that are made from the above-described antibodies and fragments thereof. In another aspect, the invention relates to a polynucleotide encoding the antibodies or fragments thereof, and to vectors comprising the polynucleotides. 
       BACKGROUND OF THE INVENTION 
       [0003]    Insulin-like growth factor-I receptor (IGF-I receptor) is a transmembrane heterotetrameric protein, which has two extracellular alpha chains and two membrane-spanning beta chains in a disulfide-linked β-α-α-β configuration. The binding of the ligands, which are insulin-like growth-factor-I (IGF-I) and insulin-like growth factor-II (IGF-II), by the extracellular domain of IGF-I receptor activates its intracellular tyrosine kinase domain resulting in autophosphorylation of the receptor and substrate phosphorylation. The IGF-I receptor is homologous to insulin receptor, having a high sequence similarity of 84% in the beta chain tyrosine kinase domain and a low sequence similarity of 48% in the alpha chain extracellular cysteine rich domain (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). The IGF-I receptor and its ligands (IGF-I and IGF-II) play important roles in numerous physiological processes including growth and development during embryogenesis, metabolism, cellular proliferation and cell differentiation in adults (LeRoith, D., 2000 , Endocrinology,  141, 1287-1288; LeRoith, D., 1997 , New England J. Med.,  336, 633-640). 
         [0004]    IGF-I and IGF-II function both as endocrine hormones in the blood, where they are predominantly present in complexes with IGF-binding proteins, and as paracrine and autocrine growth factors that are produced locally (Humbel, R. E., 1990 , Eur. J. Biochem.,  190, 445-462; Cohick, W. S. and Clemmons, D. R., 1993 , Annu. Rev. Physiol.  55, 131-153). 
         [0005]    The IGF-I receptor has been implicated in promoting growth, transformation and survival of tumor cells (Baserga, R. et al., 1997 , Biochem. Biophys. Acta,  1332, F105-F126; Blakesley, V. A. et al., 1997 , Journal of Endocrinology,  152, 339-344; Kaleko, M., Rutter, W. J., and Miller, A. D. 1990 , Mol. Cell. Biol.,  10, 464-473). Thus, several types of tumors are known to express higher than normal levels of IGF-I receptor, including breast cancer, colon cancer, ovarian carcinoma, synovial sarcoma and pancreatic cancer (Khandwala, H. M. et al., 2000 , Endocrine Reviews,  21, 215-244; Werner, H. and LeRoith, D., 1996 , Adv. Cancer Res.,  68, 183-223; Happerfield, L. C. et al., 1997 , J. Pathol.,  183, 412-417; Frier, S. et al., 1999 , Gut,  44, 704-708; van Dam, P. A. 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 shown to be potent mitogens for several human tumor cell lines such as lung cancer, breast cancer, colon cancer, osteosarcoma and cervical cancer (Ankrapp, D. P. and Bevan, D. R., 1993 , Cancer Res.,  53, 3399-3404; Cullen, K. J., 1990 , Cancer Res.,  50, 48-53; Hermanto, U. et al., 2000 , Cell Growth  &amp;  Differentiation,  11, 655-664; Guo, Y. S. et al., 1995 , J. Am. Coll. Surg.,  181, 145-154; Kappel, C. C. et al., 1994,  Cancer Res.,  54, 2803-2807; Steller, M. A. et al., 1996,  Cancer Res.,  56, 1761-1765). Several of these tumors and tumor cell lines also express high levels of IGF-I or IGF-II, which may stimulate their growth in an autocrine or paracrine manner (Quinn, K. A. et al., 1996,  J. Biol. Chem.,  271, 11477-11483). 
         [0006]    Epidemiological studies have shown a correlation of elevated plasma level of IGF-I (and lower level of IGF-binding protein-3) with increased risk for prostate cancer, colon cancer, lung cancer and breast cancer (Chan, J. M. 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, S. E. et al., 1998,  Lancet,  351, 1393-1396). Strategies to lower the IGF-I level in plasma or to inhibit the function of IGF-I receptor have been suggested 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). 
         [0007]    The IGF-I receptor protects tumor cells from apoptosis caused by growth factor deprivation, anchorage independence or cytotoxic drug treatment (Navarro, M. and Baserga, R., 2001 , Endocrinology,  142, 1073-1081; Baserga, R. et al., 1997 , Biochem. Biophys. Acta,  1332, F105-F126). The domains of IGF-I receptor that are critical for its mitogenic, transforming and anti-apoptotic activities have been identified by mutational analysis. 
         [0008]    For example, the tyrosine 1251 residue of IGF-I receptor has been identified as critical for anti-apoptotic and transformation activities but not for its mitogenic activity (O&#39;Connor, R. et al., 1997 , Mol. Cell. Biol.,  17, 427-435; Miura, M. et al., 1995,  J. Biol. Chem.,  270, 22639-22644). The intracellular signaling pathway of ligand-activated IGF-I receptor involves phosphorylation of tyrosine residues of insulin receptor substrates (IRS-1 and IRS-2), which recruit phosphatidylinositol-3-kinase (PI-3-kinase) to the membrane. The membrane-bound phospholipid products of PI-3-kinase activate a serine/threonine kinase Akt, whose substrates include the pro-apoptotic protein BAD which is phosphorylated to an inactive state (Datta, S. R., Brunet, A. and Greenberg, M. E., 1999 , Genes  &amp;  Development,  13, 2905-2927; Kulik, G., Klippel, A. and Weber, M. J., 1997 , Mol. Cell. Biol.  17, 1595-1606). The mitogenic signaling of IGF-I receptor in MCF-7 human breast cancer cells requires PI-3-kinase and is independent of mitogen-activated protein kinase, whereas the survival signaling in differentiated rat pheochromocytoma PC12 cells requires both PI-3-kinase and mitogen-activated protein kinase pathways (Dufourny, B. et al., 1997,  J. Biol. Chem.,  272, 31163-31171; Parrizas, M., Saltiel, A. R. and LeRoith, D., 1997 , J. Biol. Chem.,  272, 154-161). 
         [0009]    Down-regulation of IGF-I receptor level by anti-sense strategies has been shown to reduce the tumorigenicity of several tumor cell lines in vivo and in vitro, such as melanoma, lung carcinoma, ovarian cancer, glioblastoma, neuroblastoma and rhabdomyosarcoma (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, D. N. et al., 1994,  J. Clin. Invest.,  94, 1235-1242). Likewise, a dominant negative mutant of IGF-I receptor has been reported to reduce the tumorigenicity in vivo and growth in vitro of transformed Rat-1 cells overexpressing IGF-I receptor (Prager, D. et al., 1994 , Proc. Natl. Acad. Sci. USA,  91, 2181-2185). 
         [0010]    Tumor cells expressing an antisense to the IGF-I receptor mRNA undergo massive apoptosis when injected into animals in biodiffusion chambers. This observation makes the IGF-I receptor an attractive therapeutic target, based upon the hypothesis that tumor cells are more susceptible than normal cells to apoptosis by inhibition of IGF-I receptor (Resnicoff, M. et al., 1995,  Cancer Res.,  55, 2463-2469; Baserga, R., 1995 , Cancer Res.,  55, 249-252). 
         [0011]    Another strategy to inhibit the function of IGF-I receptor in tumor cells has been to use anti-IGF-I receptor antibodies which bind to the extracellular domains of IGF-I receptor and inhibit its activation. Several attempts have been reported to develop mouse monoclonal antibodies against IGF-I receptor, of which two inhibitory antibodies—IR3 and 1H7—are available and their use has been reported in several IGF-I receptor studies. 
         [0012]    The IR3 antibody was developed using a partially purified placental preparation of insulin receptor to immunize mice, which yielded an antibody, IR1, that was selective for binding insulin receptor, and two antibodies, IR2 and IR3, that showed preferential immunoprecipitation of IGF-I receptor (somatomedin-C receptor) but also weak immunoprecipitation of insulin receptor (Kull, F. C. et al., 1983,  J. Biol. Chem.,  258, 6561-6566). 
         [0013]    The 1H7 antibody was developed by immunizing mice with purified placental preparation of IGF-I receptor, which yielded the inhibitory antibody 1H7 in addition to three stimulatory antibodies (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). 
         [0014]    In another report, a panel of mouse monoclonal antibodies specific for human IGF-I receptor were obtained by immunization of mice with transfected 3T3 cells expressing high levels of IGF-I receptor, which were categorized into seven groups by binding competition studies and by their inhibition or stimulation of IGF-I binding to transfected 3T3 cells (Soos, M. A. et al., 1992,  J. Biol. Chem.,  267, 12955-12963). 
         [0015]    Thus, although IR3 antibody is the most commonly used inhibitory antibody for IGF-I receptor studies in vitro, it suffers from the drawback that it exhibits agonistic activity in transfected 3T3 and CHO cells expressing human IGF-I receptor (Kato, H. et al., 1993,  J. Biol. Chem.,  268, 2655-2661; Steele-Perkins, G. and Roth, R. A., 1990 , Biochem. Biophys. Res. Commun.,  171, 1244-1251). Similarly, among the panel of antibodies developed by Soos et al., the most inhibitory antibodies 24-57 and 24-60 also showed agonistic activities in the transfected 3T3 cells (Soos, M. A. et al., 1992,  J. Biol. Chem.,  267, 12955-12963). Although, IR3 antibody is reported to inhibit the binding of IGF-I (but not IGF-II) to expressed receptors in intact cells and after solubilization, it is shown to inhibit the ability of both IGF-I and IGF-II to stimulate DNA synthesis in cells in vitro (Steele-Perkins, G. and Roth, R. A., 1990 , Biochem. Biophys. Res. Commun.,  171, 1244-1251). The binding epitope of IR3 antibody has been inferred from chimeric insulin-IGF-I receptor constructs to be the 223-274 region of IGF-I receptor (Gustafson, T. A. and Rutter, W. J., 1990 , J. Biol. Chem.,  265, 18663-18667; Soos, M. A. et al., 1992,  J. Biol. Chem.,  267, 12955-12963). 
         [0016]    The MCF-7 human breast cancer cell line is typically used as a model cell line to demonstrate the growth response of IGF-I and IGF-II in vitro (Dufourny, B. et al., 1997 , J. Biol. Chem.,  272, 31163-31171). In MCF-7 cells, the IR3 antibody incompletely blocks the stimulatory effect of exogenously added IGF-I and IGF-II in serum-free conditions by approximately 80%. Also, the IR3 antibody does not significantly inhibit (less than 25%) the growth of MCF-7 cells in 10% serum (Cullen, K. J. et al., 1990,  Cancer Res.,  50, 48-53). This weak inhibition of serum-stimulated growth of MCF-7 cells by IR3 antibody in vitro may be related to the results of an in vivo study in which IR3 antibody treatment did not significantly inhibit the growth of a MCF-7 xenograft in nude mice (Arteaga, C. L. et al., 1989,  J. Clin. Invest.,  84, 1418-1423). 
         [0017]    Because of the weak agonistic activities of the IR3 and other reported antibodies, and their inability to significantly inhibit the growth of tumor cells such as MCF-7 cells in the more physiological condition of serum-stimulation (instead of stimulation by exogenously added IGF-I or IGF-II in serum-free condition), there is a need for new anti-IGF-I receptor antibodies which significantly inhibit the serum-stimulated growth of tumor cells but which do not show significant agonistic activity by themselves. 
       SUMMARY OF THE INVENTION 
       [0018]    Accordingly, it is an object of the invention to provide antibodies, antibody fragments and antibody derivatives that specifically bind to insulin-like growth factor-I receptor and inhibit the cellular activity of the receptor by antagonizing the receptor, and are also substantially devoid of agonist activity towards the receptor. 
         [0019]    Thus, in a first embodiment, there is provided murine antibody EM164, which is fully characterized herein with respect to the amino acid sequences of both its light and heavy chain variable regions, the cDNA sequences of the genes for the light and heavy chain variable regions, the identification of its CDRs (complementarity-determining regions), the identification of its surface amino acids, and means for its expression in recombinant form. 
         [0020]    In a second embodiment, there are provided resurfaced or humanized versions of antibody EM164 wherein surface-exposed residues of the antibody or its fragments are replaced in both light and heavy chains to more closely resemble known human antibody surfaces. Such humanized antibodies may have increased utility, compared to murine EM164, as therapeutic or diagnostic agents. Humanized versions of antibody EM164 are also fully characterized herein with respect to their respective amino acid sequences of both light and heavy chain variable regions, the DNA sequences of the genes for the light and heavy chain variable regions, the identification of the CDRs, the identification of their surface amino acids, and disclosure of a means for their expression in recombinant form. 
         [0021]    In a third embodiment, there is provided an antibody that is capable of inhibiting the growth of a cancer cell by greater than about 80% in the presence of a growth stimulant such as, for example, serum, insulin-like growth factor-I and insulin-like growth factor-II. 
         [0022]    In a fourth embodiment, there is provided an antibody or antibody fragment having a heavy chain including CDRs having amino acid sequences represented by SEQ ID NOS:1-3, respectively: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 SYWMH, 
                 (SEQ ID NO:1) 
                   
               
               
                   
                   
               
               
                   
                 EINPSNGRTNYNEKFKR, 
                 (SEQ ID NO:2) 
               
               
                   
                   
               
               
                   
                 GRPDYYGSSKWYFDV; 
                 (SEQ ID NO:3) 
               
             
          
         
       
     
         [0023]    and having a light chain that comprises CDRs having amino acid sequences represented by SEQ ID NOS:4-6: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 RSSQSIVHSNVNTYLE; 
                 (SEQ ID NO:4) 
                   
               
               
                   
                   
               
               
                   
                 KVSNRFS; 
                 (SEQ ID NO:5) 
               
               
                   
                   
               
               
                   
                 FQGSHVPPT. 
                 (SEQ ID NO:6) 
               
             
          
         
       
     
         [0024]    In a fifth embodiment, there are provided antibodies having a heavy chain that has an amino acid sequence that shares at least 90% sequence identity with an amino acid sequence represented by SEQ ID NO:7: 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO:7) 
                   
               
             
          
           
               
                 QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGE 
                   
               
               
                   
               
               
                 INPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGR 
               
               
                   
               
               
                 PDYYGSSKWYFDVWGAGTTVTVSS. 
               
             
          
         
       
     
         [0025]    Similarly, there are provided antibodies having a light chain that has an amino acid sequence that shares at least 90% sequence identity with an amino acid sequence represented by SEQ ID NO:8: 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO:8) 
                   
               
             
          
           
               
                 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPK 
                   
               
               
                   
               
               
                 LLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVP 
               
               
                   
               
               
                 PTFGGGTKLEIKR. 
               
             
          
         
       
     
         [0026]    In a sixth embodiment, antibodies are provided having a humanized or resurfaced light chain variable region having an amino acid sequence corresponding to one of SEQ ID NOS:-9-12: 
         [0000]    
       
         
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
               
               
             
           
               
                 (SEQ ID NO:9) 
                   
               
             
          
           
               
                 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPR 
                   
               
               
                   
               
               
                 LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP 
               
               
                   
               
               
                 PTFGGGTKLEIKR; 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO:10) 
                   
               
             
          
           
               
                 DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPK 
                   
               
               
                   
               
               
                 LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP 
               
               
                   
               
               
                 PTFGGGTKLEIKR; 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO:11) 
                   
               
             
          
           
               
                 DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPR 
                   
               
               
                   
               
               
                 LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP 
               
               
                   
               
               
                 PTFGGGTKLEIKR; 
               
               
                 or 
               
               
                   
               
             
          
           
               
                 (SEQ ID NO:12) 
                   
               
             
          
           
               
                 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPK 
                   
               
               
                   
               
               
                 LLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP 
               
               
                   
               
               
                 PTFGGGTKLEIKR. 
               
             
          
         
       
     
         [0027]    Similarly, antibodies are provided having a humanized or resurfaced heavy chain variable region having an amino acid sequence corresponding to SEQ ID NO:13: 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO:13) 
                   
               
             
          
           
               
                 QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGE 
                   
               
               
                   
               
               
                 INPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGR 
               
               
                   
               
               
                 PDYYGSSKWYFDVWGQGTTVTVSS. 
               
             
          
         
       
     
         [0028]    In a seventh embodiment, antibodies or antibody fragments of the present invention are provided that have improved properties. For example, antibodies or antibody fragments having improved affinity for IGF-I-receptor are prepared by affinity maturation of an antibody or fragment of the present invention. 
         [0029]    The present invention further provides conjugates of said antibodies, wherein a cytotoxic agent is covalently attached, directly or via a cleavable or non-cleavable linker, to an antibody or epitope-binding fragment of an antibody of the present invention. In preferred embodiments, the cytotoxic agent is a taxol, a maytansinoid, CC-1065 or a CC-1065 analog. 
         [0030]    The present invention further provides for antibodies or fragments thereof that are further labeled for use in research or diagnostic applications. In preferred embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent or a metal ion. 
         [0031]    A method for diagnosis is also provided in which said labeled antibodies or fragments are administered to a subject suspected of having a cancer, and the distribution of the label within the body of the subject is measured or monitored. 
         [0032]    In a eighth embodiment, the invention provides methods for the treatment of a subject having a cancer by administering an antibody, antibody fragment or antibody conjugate of the present invention, either alone or in combination with other cytotoxic or therapeutic agents. The cancer can be one or more of, for example, breast cancer, colon cancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovial carcinoma, pancreatic cancer, or other cancer yet to be determined in which IGF-I receptor levels are elevated. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0033]      FIG. 1  shows fluorescence activated cell sorting (FACS) analysis of the specific binding of purified EM164 antibody to cells overexpressing human Y1251F IGF-I receptor or human insulin receptor. 
           [0034]      FIG. 2  shows a binding titration curve for the binding of EM164 antibody to biotinylated human IGF-I receptor. 
           [0035]      FIG. 3  shows the inhibition of the binding of biotinylated IGF-I to human breast cancer MCF-7 cells by EM164 antibody. 
           [0036]      FIG. 4  shows the inhibition of IGF-I-stimulated autophosphorylation of IGF-I receptor in MCF-7 cells by EM164 antibody. 
           [0037]      FIG. 5  shows the inhibition of IGF-I-stimulated IRS-1-phosphorylation in MCF-7 cells by EM164 antibody. 
           [0038]      FIG. 6  shows the inhibition of IGF-I-stimulated signal transduction in SaOS-2 cells by EM164 antibody. 
           [0039]      FIG. 7  shows the effect of EM164 antibody on the growth and survival of MCF-7 cells under different growth conditions, as assessed by MTT assay. 
           [0040]      FIG. 8  shows the effect of EM164 antibody on the growth and survival of MCF-7 cells in the presence of various serum concentrations. 
           [0041]      FIG. 9  shows the inhibition of IGF-1- and serum-stimulated growth and survival of NCI-H838 cells by EM164 antibody. 
           [0042]      FIG. 10  shows the effect of treatment with EM164 antibody, taxol, or a combination of EM164 antibody and taxol, on the growth of a Calu-6 lung cancer xenograft in mice. 
           [0043]      FIG. 11  shows competition between the binding of humanized EM164 antibody (v.1.0) and murine EM164 antibody. 
           [0044]      FIG. 12  shows the cDNA (SEQ ID NO:49) and amino acid sequences (SEQ ID NO:50) of the light chain leader and variable region of the murine anti-IGF-I receptor antibody EM164. The arrow marks the start of framework 1. The 3 CDR sequences according to Kabat are underlined. 
           [0045]      FIG. 13  shows the cDNA (SEQ ID NO:51) and amino acid sequences (SEQ ID NO:52) of the heavy chain leader and variable region for the murine anti-IGF-I receptor antibody EM164. The arrow marks the start of framework 1. The 3 CDR sequences according to Kabat are underlined. 
           [0046]      FIG. 14  shows the light and heavy chain CDR amino acid sequences of antibody EM164 as determined from Chothia canonical class definitions. AbM modeling software definitions for the heavy chain CDRs are also shown. Light Chain: CDR1 is SEQ ID NO:4, CDR2 is SEQ ID NO:5, and CDR3 is 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. 
           [0047]      FIG. 15  shows the light chain and heavy chain amino acid sequences for anti-IGF-I-receptor antibody EM164 aligned with the germline sequences for the Cr1 (SEQ ID NO:56) and J558.c genes (SEQ ID NO:57). Dashes (−) indicate sequence identity. 
           [0048]      FIG. 16  shows the plasmids used to build and express the recombinant chimeric and humanized EM164 antibodies. A) a light chain cloning plasmid, B) a heavy chain cloning plasmid, C) a mammalian antibody expression plasmid. 
           [0049]      FIG. 17  shows the 10 most homologous amino acid sequences of the light chains screened from the 127 antibodies in the set of structure files used to predict the surface residues 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 (SE 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). 
           [0050]      FIG. 18  shows the 10 most homologous amino acid sequences of the heavy chains screened from the 127 antibodies in the set of structure files used to predict the surface residues of EM164. em164 HC (SEQ ID NO:70), 1nqb (SEQ ID NO:71), 1ngp (SEQ ID NO:72), 1fbi (SEQ ID NO:73), 1afy (SEQ ID NO:74), 1yuh (SE 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). 
           [0051]      FIG. 19  shows the average accessibility for each of the (A) light, and (B) heavy chain variable region residues from the 10 most homologous structures. The numbers represent the Kabat antibody sequence position numbers. 
           [0052]      FIG. 20  shows the light chain variable region amino acid sequences for murine EM164 (muEM164) and humanized EM164 (huEM164) antibodies. muEM164 (SEQ ID NO:82), huEM164 V1.0 (SEQ ID NO:83), huEM164 V1.1 (SEQ ID NO:84), huEM164 V1.2 (SEQ ID NO:85), huEM164 V1.3 (SEQ ID NO:86). 
           [0053]      FIG. 21  shows the heavy chain variable region amino acid sequences for murine (muEM164, SEQ ID NO:87) and humanized EM164 antibodies (huEM164, SEQ ID NO:88). 
           [0054]      FIG. 22  shows the huEM164 v1.0 variable region DNA and amino acid sequences for both the light (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). 
           [0055]      FIG. 23  shows the light chain variable region DNA and amino acid sequences for 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; amino acid SEQ ID NO:98). 
           [0056]      FIG. 24  shows the inhibition of IGF-I-stimulated growth and survival of MCF-7 cells by humanized EM164 v1.0 antibody and murine EM164 antibody. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0057]    The present inventors have discovered and improved novel antibodies that specifically bind to the human insulin-like growth factor-I receptor (IGF-IR) on the cell surface. The antibodies and fragments have the unique ability to inhibit the cellular functions of the receptor without the capacity to activate the receptor themselves. Thus, while previously known antibodies that specifically bind and inhibit IGF-IR also activate the receptor even in the absence of IGF-IR ligands, the antibodies or fragments of the present invention antagonize IGF-IR but are substantially devoid of agonist activity. Furthermore, the antibodies and antibody fragments of the present invention inhibit the growth of human tumor cells such as MCF-7 cells in the presence of serum by greater than 80%, which is a higher degree of inhibition than is obtained using previously known anti-IGF-IR antibodies. 
         [0058]    The present invention proceeds from a murine anti-IGF-IR antibody, herein EM164, that is fully characterized with respect to the amino acid sequences of both light and heavy chains, the identification of the CDRs, the identification of surface amino acids, and means for its expression in recombinant form. 
         [0059]    The germline sequences are shown in  FIG. 15  aligned with the sequence of EM164. The comparison identifies probable somatic mutations in EM164, including one each in CDR1 in the light chain and in CDR2 in the heavy chain. 
         [0060]    The primary amino acid and DNA sequences of antibody EM164 light and heavy chains, and of humanized versions, are disclosed herein. However, the scope of the present invention is not limited to antibodies and fragments comprising these sequences. Instead, all antibodies and fragments that specifically bind to an insulin-like growth factor-I receptor and antagonize the biological activity of the receptor, but which are substantially devoid of agonist activity, fall within the scope of the present invention. Thus, antibodies and antibody fragments may differ from antibody EM164 or the humanized derivatives in the amino acid sequences of their scaffold, CDRs, light chain and heavy chain, and still fall within the scope of the present invention. 
         [0061]    The CDRs of antibody EM164 are identified by modeling and their molecular structures have been predicted. Again, while the CDRs are important for epitope recognition, they are not essential to the antibodies and fragments of the invention. Accordingly, antibodies and fragments are provided that have improved properties produced by, for example, affinity maturation of an antibody of the present invention. 
         [0062]    Diverse antibodies and antibody fragments, as well as antibody mimics may be readily produced by mutation, deletion and/or insertion within the variable and constant region sequences that flank a particular set of CDRs. Thus, for example, different classes of Ab are possible for a given set of CDRs by substitution of different heavy chains, whereby, for example, IgG1-4, IgM, IgA1-2, IgD, IgE antibody types and isotypes may be produced. Similarly, artificial antibodies within the scope of the invention may be produced by embedding a given set of CDRs within an entirely synthetic framework. The term “variable” is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its antigen. However, the variability is not usually evenly distributed through the variable domains of the antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of heavy and light chains each comprise four framework regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (E. A. Kabat et al.  Sequences of Proteins of Immunological Interest , fifth edition, 1991, NIH). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. 
         [0063]    Humanized antibodies, or antibodies adapted for non-rejection by other mammals, may be produced using several technologies such as resurfacing and CDR grafting. In the resurfacing technology, molecular modeling, statistical analysis and mutagenesis are combined to adjust the non-CDR surfaces of variable regions to resemble the surfaces of known antibodies of the target host. Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed in U.S. Pat. No. 5,639,641, which is hereby incorporated in its entirety by reference. In the CDR grafting technology, the murine heavy and light chain CDRs are grafted into a fully human framework sequence. 
         [0064]    The invention also includes functional equivalents of the antibodies described in this specification. Functional equivalents have binding characteristics that are comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239,400; PCT Application WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424, which are incorporated in their respective entireties by reference. 
         [0065]    Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. “Substantially the same” as applied to an amino acid sequence is defined herein as a sequence with at least about 90%, and more preferably at least about 95% sequence identity to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman,  Proc. Natl. Acad. Sci. USA  85, 2444-2448 (1988). 
         [0066]    Chimerized antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human. Humanized forms of the antibodies are made by substituting the complementarity determining regions of, for example, a mouse antibody, into a human framework domain, e.g., see PCT Pub. No. W092/22653. Humanized antibodies preferably have constant regions and variable regions other than the complementarity determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human. 
         [0067]    Functional equivalents also include single-chain antibody fragments, also known as single-chain antibodies (scFvs). These fragments contain at least one fragment of an antibody variable heavy-chain amino acid sequence (V H ) tethered to at least one fragment of an antibody variable light-chain sequence (V L ) with or without one or more interconnecting linkers. Such a linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the (V L ) and (V H ) domains occurs once they are linked so as to maintain the target molecule binding-specificity of the whole antibody from which the single-chain antibody fragment is derived. Generally, the carboxyl terminus of the (V L ) or (V H ) sequence may be covalently linked by such a peptide linker to the amino acid terminus of a complementary (V L ) and (V H ) sequence. Single-chain antibody fragments may be generated by molecular cloning, antibody phage display library or similar techniques. These proteins may be produced either in eukaryotic cells or prokaryotic cells, including bacteria. 
         [0068]    Single-chain antibody fragments contain amino acid sequences having at least one of the variable or complementarity determining regions (CDRs) of the whole antibodies described in this specification, but are lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of whole antibodies. Single-chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing a part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity. Additionally, single-chain antibody fragments are considerably smaller than whole antibodies and may therefore have greater capillary permeability than whole antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Also, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely to provoke an immune response in a recipient than whole antibodies. 
         [0069]    Functional equivalents further include fragments of antibodies that have the same, or comparable binding characteristics to those of the whole antibody. Such fragments may contain one or both Fab fragments or the F(ab′) 2  fragment. Preferably the antibody fragments contain all six complementarity determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five CDRs, are also functional. Further, the functional equivalents may be or may combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof. 
         [0070]    The knowledge of the amino acid and nucleic acid sequences for the anti-IGF-I receptor antibody EM164 and its humanized variants, which are described herein, can be used to develop other antibodies which also bind to human IGF-I receptor and inhibit the cellular functions of the IGF-I receptor. Several studies have surveyed the effects of introducing one or more amino acid changes at various positions in the sequence of an antibody, based on the knowledge of the primary antibody sequence, on its properties such as binding and level of expression (Yang, W. P. et al., 1995,  J. Mol. Biol.,  254, 392-403; Rader, C. et al., 1998 , Proc. Natl. Acad. Sci. USA,  95, 8910-8915; Vaughan, T. J. et al., 1998,  Nature Biotechnology,  16, 535-539). 
         [0071]    In these studies, variants of the primary antibody have been generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2, CDR3, or framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of  E. coli  (Vaughan, T. J. et al., 1998,  Nature Biotechnology,  16, 535-539; Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “ Phage Display of Peptides and Proteins ”, Eds. Kay, B. K. et al., Academic Press). These methods of changing the sequence of the primary antibody have resulted in improved affinities of the secondary antibodies (Gram, H. et al., 1992 , Proc. Natl. Acad. Sci. USA,  89, 3576-3580; Boder, E. T. et al., 2000 , Proc. Natl. Acad. Sci. USA,  97, 10701-10705; Davies, J. and Riechmann, L., 1996 , Immunotechnology,  2, 169-179; Thompson, J. et al., 1996,  J. Mol. Biol.,  256, 77-88; Short, M. K. et al., 2002,  J. Biol. Chem.,  277, 16365-16370; Furukawa, K. et al., 2001,  J. Biol. Chem.,  276, 27622-27628). 
         [0072]    By a similar directed strategy of changing one or more amino acid residues of the antibody, the antibody sequences described in this invention can be used to develop anti-IGF-I receptor antibodies with improved functions. 
         [0073]    The conjugates of the present invention comprise the antibody, fragments, and their analogs as disclosed herein, linked to a cytotoxic agent. Preferred cytotoxic agents are maytansinoids, taxanes and analogs of CC-1065. The conjugates can be prepared by in vitro methods. In order to link the cytotoxic agent to the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Preferred linking groups are disulfide groups and thioether groups. For example, conjugates can be constructed using a disulfide exchange reaction or by forming a thioether bond between the antibody and the cytotoxic agent. 
         [0074]    Maytansinoids and maytansinoid analogs are among the preferred cytotoxic agents. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat. Nos. 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. 
         [0075]    Taxanes are also preferred cytotoxic agents. Taxanes suitable for use in the present invention are disclosed in U.S. Pat. Nos. 6,372,738 and 6,340,701. 
         [0076]    CC-1065 and its analogs are also preferred cytotoxic drugs for use in the present invention. CC-1065 and its analogs are disclosed in U.S. Pat. Nos. 6,372,738; 6,340,701; 5,846,545 and 5,585,499. 
         [0077]    An attractive candidate for the preparation of such cytotoxic conjugates is CC-1065, which is a potent anti-tumor antibiotic isolated from the culture broth of  Streptomyces zelensis . CC-1065 is about 1000-fold more potent in vitro than are commonly used anti-cancer drugs, such as doxorubicin, methotrexate and vincristine (B. K. Bhuyan et al.,  Cancer Res.,  42, 3532-3537 (1982)). 
         [0078]    Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and calicheamicin are also suitable for the preparation of conjugates of the present invention, and the drug molecules can also be linked to the antibody molecules through an intermediary carrier molecule such as serum albumin. 
         [0079]    For diagnostic applications, the antibodies of the present invention typically will be labeled with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as  3 H,  14 C,  32 P,  35 S, or  131 I; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. 
         [0080]    Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by 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.  30:407 (1982). 
         [0081]    The antibodies of the present invention can be employed in any known assay method, 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)). 
         [0082]    The antibodies of the invention also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety such as a radio-opaque agent or radioisotope is administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of malignancies. The antibody may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art. 
         [0083]    The antibodies of the invention also are useful as affinity purification agents. In this process, the antibodies are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. 
         [0084]    The antibodies of the invention also are useful as reagents in biological research, based on their inhibition of the function of IGF-I receptor in cells. 
         [0085]    For therapeutic applications, the antibodies or conjugates of the invention are administered to a subject, in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibody may also be administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those of skill in the art as the clinical situation warrants. Examples of suitable carriers, diluents and/or excipients include: (1) Dulbecco&#39;s phosphate buffered saline, pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The method of the present invention can be practiced in vitro, in vivo, or ex vivo. 
         [0086]    In other therapeutic treatments, the antibodies, antibody fragments or conjugates of the invention are co-administered with one or more additional therapeutic agents. Suitable therapeutic agents include, but are not limited to, cytotoxic or cytostatic agents. Taxol is a preferred therapeutic agent that is also a cytotoxic agent. 
         [0087]    Cancer therapeutic agents are those agents that seek to kill or limit the growth of cancer cells while doing minimal damage to the host. Thus, such agents may exploit any difference in cancer cell properties (e.g. metabolism, vascularization or cell-surface antigen presentation) from healthy host cells. Differences in tumor morphology are potential sites for intervention: for example, the second therapeutic can be an antibody such as an anti-VEGF antibody that is useful in retarding the vascularization of the interior of a solid tumor, thereby slowing its growth rate. Other therapeutic agents include, but are not limited to, adjuncts such as granisetron HCL, androgen inhibitors such as leuprolide acetate, antibiotics such as doxorubicin, antiestrogens such as tamoxifen, antimetabolites such as interferon alpha-2a, cytotoxic agents such as taxol, enzyme inhibitors such as ras farnesyl-transferase inhibitor, immunomodulators such as aldesleukin, and nitrogen mustard derivatives such as melphalan HCl, and the like. 
         [0088]    When present in an aqueous dosage form, rather than being lyophilized, the antibody typically will be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml, although wide variation outside of these ranges is permitted. For the treatment of disease, the appropriate dosage of antibody or conjugate will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibodies are administered for preventive or therapeutic purposes, the course of previous therapy, the patient&#39;s clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. 
         [0089]    Depending on the type and severity of the disease, about 0.015 to 15 mg of antibody/kg of patient weight is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are not excluded. 
       EXAMPLES 
       [0090]    The invention is now described by reference to the following examples, which are illustrative only, and are not intended to limit the present invention. 
       Example 1 
     Murine EM164 Antibody 
       [0091]    In this first example, the complete primary amino acid structure and cDNA sequence of a murine antibody of the present invention is disclosed, together with its binding properties and means for its expression in recombinant form. Accordingly, there is provided a full and complete disclosure of an antibody of the invention and its preparation, such that one of ordinary skill in the immunological arts would be able to prepare said antibody without undue experimentation. 
       A. Generation of Anti-IGF-I Receptor Monoclonal Antibody Hybridoma 
       [0092]    A cell line expressing human IGF-I receptor with a Y1251F mutation was used for immunization as it expressed a high number of IGF-I receptors (˜10 7  per cell). The Y1251F-mutation in the cytoplasmic domain of IGF-I receptor resulted in loss of transformation and anti-apoptotic signaling, but did not affect IGF-I binding and IGF-I-stimulated mitogenic signaling (O&#39;Connor, R. et al., 1997 , Mol. Cell. Biol.,  17, 427-435; Miura, M. et al., 1995,  J. Biol. Chem.,  270, 22639-22644). The mutation did not otherwise affect antibody generation because the antibody of this example bound to the extracellular domain of IGF-I receptor, which was identical for both the Y1251F mutant and the wild type receptor. 
         [0093]    A cell line expressing human IGF-I receptor with a Y1251F mutation was generated from 3T3-like cells of a IGF-I-receptor-deficient mouse by transfection with Y1251F-mutant human IGF-I-receptor gene together with a puromycin-resistance gene, and was selected using puromycin (2.5 microgram/mL) and by FACS sorting for high IGF-I receptor expression (Miura, M. et al., 1995 , J. Biol. Chem.,  270, 22639-22644). A cell line having a high level of IGF-I receptor expression was further selected using a high concentration of puromycin such as 25 microgram/mL, which was toxic to most of the cells. Surviving colonies were picked and those displaying a high level of IGF-I receptor expression were selected. 
         [0094]    CAF1/J female mice, 6 months old, 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). The animals were boosted with 0.2 mL cell suspension as follows: day 2, 1×10 6  cells; day 5, 2×10 6  cells; days 7, 9, 12, and 23, 1×10 7  cells. On day 26, a mouse was sacrificed and its spleen removed. 
         [0095]    The spleen was ground between two frosted glass slides to obtain a single cell suspension, which was washed with serum-free RPMI medium containing penicillin and streptomycin (SFM). The spleen cell pellet was resuspended in 10 mL of 0.83% (w/v) ammonium chloride solution in water for 10 min on ice to lyse the red blood cells, and was then washed with serum-free medium (SFM). Spleen cells (1.2×10 8 ) were pooled with myeloma cells (4×10 7 ) from the non-secreting mouse myeloma cell line P3X63Ag8.653 (ATCC, Rockville, Md.; Cat. # CRL1580) in a tube, and washed with the serum-free RPMI-1640 medium (SFM). The supernatant was removed and the cell pellet resuspended in the residual medium. The tube was placed in a beaker of water at 37° C. 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 drop rate of 0.5 mL/minute while the tube was gently shaken. After a wait of one minute, 10 mL of SFM was added as follows: 1 mL over the first minute, 2 mL over the second minute, and 7 mL over the third minute. Another 10 mL was then added slowly over one minute. Cells were pelleted by centrifugation, washed in SFM and resuspended in RPMI-1640 growth medium supplemented with 5% fetal bovine serum (FBS), hypoxanthine/aminopterin/thymidine (HAT), penicillin, streptomycin, and 10% hybridoma cloning supplement (HCS). Cells were seeded into 96-well flat-bottom tissue culture plates at 2×10 5  spleen cells in 200 μL per well. After 5-7 days, 100 μL per well were removed and replaced with growth medium supplemented with hypoxanthine/thymidine (HT) and 5% FBS. The general conditions used for immunization and hybridoma production were as described by 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 techniques of immunization and hybridoma production can also be used, as are well known to those of skill in the art. 
         [0096]    Culture supernatants from hybridoma clones were screened for binding to purified human IGF-I receptor by ELISA, for specific binding to cells overexpressing human IGF-I receptor, and for a lack of binding to cells overexpressing human insulin receptor by ELISA and FACS screening as described below. Clones exhibiting higher binding affinity to cells overexpressing human IGF-I receptor than to cells overexpressing human insulin receptor were expanded and subcloned. The culture supernatants of the subclones were further screened by the above binding assays. By this procedure, subclone 3F1-C8-D7 (EM164) was selected, and the heavy and light chain genes were cloned and sequenced as described below. 
         [0097]    Human IGF-I receptor was isolated for use in the screening of supernatants from hybridoma clones for their binding to IGF-I receptor by the method below. Biotinylated IGF-I was prepared by modification of recombinant IGF-I using biotinylating reagents such as sulfo-NHS-LC-biotin, sulfo-NHS-SS-biotin, or NHS-PEO 4 -biotin. Biotinylated IGF-I was absorbed on streptavidin-agarose beads and incubated with lysate from cells that overexpressed human wild type or Y1251F mutant IGFR. The beads were washed and eluted with a buffer containing 2 to 4 M urea and detergent such as triton X-100 or octyl-β-glucoside. Eluted IGF-I receptor was dialyzed against PBS and was analyzed for purity by SDS-PAGE under reducing conditions, which showed alpha and beta chain bands of IGF-I receptor of molecular weights about 135 kDa and 95 kDa, respectively. 
         [0098]    To check for the binding of hybridoma supernatants to purified IGF-I receptor, an Immulon-4HB ELISA plate (Dynatech) was coated with a purified human IGF-I receptor sample (prepared by dialysis from urea/octyl-β-glucoside elution of affinity purified sample) diluted in 50 mM CHES buffer at pH 9.5 (100 μL; 4° C., overnight). The wells were blocked with 200 μL of blocking buffer (10 mg/mL BSA in TBS-T buffer containing 50 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% tween-20) and incubated with supernatants from hybridoma clones (100 μL; diluted in blocking buffer) for about 1 h to 12 h, washed with TBS-T buffer, and incubated with goat-anti-mouse-IgG-Fc-antibody-horseradish peroxidase (HRP) conjugate (100 μL; 0.8 μg/mL in blocking buffer; Jackson ImmunoResearch Laboratories), followed by washes and detection using ABTS/H 2 O 2  substrate at 405 nm (0.5 mg/mL ABTS, 0.03% H 2 O 2  in 0.1 M citrate buffer, pH 4.2). Typically, a supernatant from a 3F1 hybridoma subclone yielded a signal of about 1.2 absorbance units within 3 min of development, in contrast to values of 0.0 obtained for supernatants from some other hybridoma clones. General conditions for this ELISA were similar to the 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), which conditions can also be used. 
         [0099]    Screening of hybridoma supernatants for specific binding to human IGF-I receptor and not to human insulin receptor was performed using ELISA on cell lines that overexpressed human Y1251F-IGF-I receptor and on cell lines that overexpressed human insulin receptor. Both cell lines were generated from 3T3-like cells of IGF-I receptor deficient mice. The IGF-I receptor overexpressing cells and insulin receptor overexpressing cells were separately harvested from tissue culture flasks by quick trypsin/EDTA treatment, suspended in growth medium containing 10% FBS, pelleted by centrifugation, and washed with PBS. The washed cells (100 μL of about 1-3×10 6  cells/mL) were added to wells of an Immulon-2HB plate coated with phytohemagglutinin (100 μL of 20 μg/mL PHA), centrifuged and allowed to adhere to PHA-coated wells for 10 min. The plate with cells was flicked to remove PBS and was then dried overnight at 37° C. The wells were blocked with 5 mg/mL BSA solution in PBS for 1 h at 37° C. and were then washed gently with PBS. Aliquots of the supernatants from hybridoma clones (100 μL; diluted in blocking buffer) were then added to wells containing IGF-I-receptor-overexpressing cells and to wells containing insulin receptor-overexpressing cells and were incubated at ambient temperature for 1 h. The wells were washed with PBS, incubated with goat-anti-mouse-IgG-Fc-antibody-horseradish peroxidase conjugate (100 μL; 0.8 μg/mL in blocking buffer) for 1 h, followed by washes and then binding was detected using an ABTS/H 2 O 2  substrate. A typical supernatant from a 3F1 hybridoma subclone upon incubation with cells overexpressing IGF-I receptor yielded a signal of 0.88 absorbance units within 12 min of development, in contrast to a value of 0.22 absorbance units obtained upon incubation with cells overexpressing human insulin receptor. 
         [0100]    The hybridoma was grown in Integra CL 350 flasks (Integra Biosciences, Maryland), according to manufacturer&#39;s specifications, to provide purified EM164 antibody. A yield of about 0.5-1 mg/mL antibody was obtained in the harvested supernatants from the Integra flasks, based on quantitation by ELISA and by SDS-PAGE/Coomassie blue staining using antibody standards. The antibody was purified by affinity chromatography on Protein A-agarose bead column under standard purification conditions of loading and washing in 100 mM Tris buffer, pH 8.9, containing 3 M NaCl, followed by elution in 100 mM acetic acid solution containing 150 mM NaCl. The eluted fractions containing antibody were neutralized with cold 2 M K 2 HPO 4  solution and dialyzed in PBS at 4° C. The concentration of the antibody was determined by measuring absorbance at 280 nm (extinction coefficient=1.4 mg −1  mL cm −1 ). The purified antibody sample was analyzed by SDS-PAGE under reducing conditions and Coomassie blue staining, which indicated only heavy and light chain bands of antibody at about 55 kDa and 25 kDa, respectively. The isotype of the purified antibody was IgG 1  with kappa light chain. 
       B. Binding Characterization of EM164 Antibody 
       [0101]    The specific binding of the purified EM164 antibody was demonstrated by fluorescence activated cell sorting (FACS) using cells overexpressing human IGF-I receptor and by using cells that overexpressed human insulin receptor ( FIG. 1 ). Incubation of EM164 antibody (50-100 nM) in 100 μL cold FACS buffer (1 mg/mL BSA in Dulbecco&#39;s MEM medium) was performed using cells overexpressing IGF-I receptor and using cells overexpressing insulin receptor (2×10 5  cells/mL) in a round-bottom 96-well plate for 1 h. The cells were pelleted by centrifugation and washed with cold FACS buffer by gentle flicking, followed by incubation with goat-anti-mouse-IgG-antibody-FITC conjugate (100 μL; 10 μg/mL in FACS buffer) on ice for 1 h. The cells were pelleted, washed, and resuspended in 120 μL of 1% formaldehyde solution in PBS. The plate was analyzed using a FACSCalibur reader (BD Biosciences). 
         [0102]    A strong fluorescence shift was obtained upon incubation of IGF-I receptor overexpressing cells with EM164 antibody, in contrast to an insignificant shift upon incubation of insulin receptor overexpressing cells with EM164 antibody ( FIG. 1 ), which demonstrated that the EM 164 antibody was selective in its binding to IGF-I receptor and did not bind to insulin receptor. The control antibodies, anti-IGF-I receptor antibody 1H7 (Santa Cruz Biotechnology) and anti-insulin receptor alpha antibody (BD Pharmingen Laboratories), yielded fluorescence shifts upon incubations with cells that overexpressed IGF-I receptor and insulin receptor, respectively ( FIG. 1 ). A strong fluorescence shift was also observed by FACS assay using EM164 antibody and human breast cancer MCF-7 cells, which expressed IGF-I receptor (Dufourny, B. et al., 1997,  J. Biol. Chem.,  272, 31163-31171), which showed that EM164 antibody bound to human IGF-I receptor on the surface of human tumor cells. 
         [0103]    The dissociation constant (K d ) for the binding of EM164 antibody with human IGF-I receptor was determined by ELISA titration of the binding of antibody at several concentrations with either directly coated IGF-I receptor (affinity purified using biotinylated IGF-I, as above) or indirectly captured biotinylated IGF-I receptor. Biotinylated IGF-I receptor was prepared by biotinylation of detergent-solubilized lysate from IGF-I receptor overexpressing cells using PEO-maleimide-biotin reagent (Pierce, Molecular Biosciences), which was affinity purified using an anti-IGF-I receptor beta chain antibody immobilized on NHS-agarose beads and was eluted with 2-4 M urea in buffer containing NP-40 detergent and dialyzed in PBS. 
         [0104]    The K d  determination for the binding of EM164 antibody with biotinylated IGF-I receptor was carried out by coating Immulon-2HB plates with 100 μL of 1 μg/mL streptavidin in carbonate buffer (150 mM sodium carbonate, 350 mM sodium bicarbonate) at 4° C. overnight. The streptavidin-coated wells were blocked with 200 μL of blocking buffer (10 mg/mL BSA in TBS-T buffer), washed with TBS-T buffer and incubated with biotinylated IGF-I receptor (10 to 100 ng) for 4 h at ambient temperature. The wells containing indirectly captured biotinylated IGF-I receptor were then washed and incubated with EM164 antibody in blocking buffer at several concentrations (5.1×10 −13  M to 200 nM) for 2 h at ambient temperature and were then incubated overnight at 4° C. The wells were next washed with TBS-T buffer and incubated with goat-anti-mouse-IgG H+L -antibody-horseradish peroxidase conjugate (100 μL; 0.5 μg/mL in blocking buffer), followed by washes and detection using ABTS/H 2 O 2  substrate at 405 nm. The value of K d  was estimated by non-linear regression for one-site binding. 
         [0105]    A similar binding titration was carried out using the Fab fragment of EM164 antibody, prepared by papain digestion of the antibody as described by E. Harlow and D. Lane (“Using Antibodies: A Laboratory Manual”; 1999, Cold Spring Harbor Laboratory Press, New York). 
         [0106]    The binding titration curve for the binding of EM164 antibody to biotinylated human IGF-I receptor yielded a K d  value of 0.1 nM ( FIG. 2 ). The Fab fragment of EM164 antibody also bound the human IGF-I receptor very tightly with a K d  value of 0.3 nM, which indicated that the monomeric binding of the EM164 antibody to IGF-I receptor was also very strong. 
         [0107]    This extremely low value of dissociation constant for the binding of IGF-I receptor by EM164 antibody was in part due to a very slow k off  rate as verified by the strong binding signals observed after prolonged 1-2 day washes of the antibody bound to immobilized IGF-I receptor. 
         [0108]    EM164 antibody can be used for immunoprecipitation of IGF-I receptor, as demonstrated by incubation of detergent-solubilized lysate of the human breast cancer MCF-7 cells with EM164 antibody immobilized on protein G-agarose beads (Pierce Chemical Company). A Western blot of the EM164 antibody immunoprecipitate was detected using a rabbit polyclonal anti-IGF-I receptor beta chain (C-terminus) antibody (Santa Cruz Biotechnology) and a goat-anti-rabbit-IgG-antibody-horseradish peroxidase conjugate, followed by washes and enhanced chemiluminescence (ECL) detection. The Western blot of EM164 immunoprecipitate from MCF-7 cells exhibited bands corresponding to the beta chain of IGF-I receptor at about 95 kDa and the pro-IGF-I receptor at about 220 kDa. Similar immunoprecipitations were carried out for other cell types to check species specificity of the binding of EM164 antibody, which also bound to IGF-I receptor from cos-7 cells (African green monkey), but did not bind to IGF-I receptor of 3T3 cells (mouse), CHO cells (chinese hamster) or goat fibroblast cells (goat). The EM164 antibody did not detect SDS-denatured human IGF-I receptor in Western blots of lysates from MCF-7 cells, which indicated that it bound to a conformational epitope of native, non-denatured human IGF-I receptor. 
         [0109]    The binding domain of EM164 antibody was further characterized using a truncated alpha chain construct, which comprised the cysteine rich domain flanked by L1 and L2 domains (residues 1-468) fused with the 16-mer-C-terminus piece (residues 704-719) and which was terminated by a C-terminus epitope tag. This smaller IGF-I receptor, which lacked residues 469-703, has been reported to bind IGF-I, although less tightly compared to the native full-length IGF-I receptor (Molina, L. et al., 2000 , FEBS Letters,  467, 226-230; Kristensen, C. et al., 1999,  J. Biol. Chem.,  274, 37251-37356). Thus, a truncated IGF-I receptor alpha chain construct was prepared comprising residues 1-468 fused to the C-terminus piece that is residues 704-719 and flanked by a C-terminus myc epitope tag. A stable cell line which expressed this construct, and which also expresses the construct transiently in human embryonic kidney 293T cells, was constructed. A strong binding of EM164 antibody to this truncated IGF-I receptor alpha chain construct was observed. Of the two antibodies tested, IR3 (Calbiochem) also bound to this truncated alpha chain, but 1H7 antibody (Santa Cruz Biotechnology) did not bind, which indicated that the epitope of EM164 antibody was clearly distinct from that of 1H7 antibody. 
       C. Inhibition of Binding of IGF-I to MCF-7 Cells by EM164 Antibody 
       [0110]    The binding of IGF-I to human breast cancer MCF-7 cells was inhibited by EM164 antibody ( FIG. 3 ). MCF-7 cells were incubated with or without 5 μg/mL EM164 antibody for 2 h in serum-free medium, followed by incubation with 50 ng/mL biotinylated IGF-I for 20 min at 37° C. The cells were then washed twice with serum-free medium to remove unbound biotin-IGF-I, and were then lysed in 50 mM HEPES, pH 7.4, containing 1% NP-40 and protease inhibitors. An Immulon-2HB ELISA plate was coated with a mouse monoclonal anti-IGF-I receptor beta chain antibody and was used to capture the IGF-I receptor and bound biotin-IGF-I from the lysate. The binding of the coated antibody to the cytoplasmic C-terminal domain of the beta chain of IGF-I receptor did not interfere with the binding of biotin-IGF-I to the extracellular domain of IGF-I receptor. The wells were washed, incubated with streptavidin-horseradish peroxidase conjugate, washed again, and then detected using ABTS/H 2 O 2  substrate. The inhibition of IGF-I binding to MCF-7 cells by 5 μg/mL EM164 antibody was essentially quantitative, and was almost equivalent to that of the ELISA background obtained using a control lacking biotin-IGF-I. 
       D. Inhibition of IGF-I Receptor Mediated Cell Signaling by EM164 Antibody 
       [0111]    Treatment of breast cancer MCF-7 cells and osteosarcoma SaOS-2 cells with EM164 antibody almost completely inhibited intracellular IGF-I receptor signaling, as shown by the inhibition of IGF-I receptor autophosphorylation and by the inhibition of phosphorylation of its downstream effectors such as insulin receptor substrate-1 (IRS-1), Akt and Erk1/2 ( FIGS. 4-6 ). 
         [0112]    In  FIG. 4 , the MCF-7 cells were grown in a 12-well plate in regular medium for 3 days, and were then treated with 20 μg/mL EM164 antibody (or anti-B4 control antibody) in serum-free medium for 3 h, followed by stimulation with 50 ng/mL IGF-I for 20 min at 37° C. The cells were then lysed in ice-cold lysis buffer containing protease and phosphatase inhibitors (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2.5 mM EDTA, 10 μM leupeptin, 5 μM pepstatin, 1 mM PMSF, 5 mM benzamidine, and 5 μg/mL aprotinin). An ELISA plate was pre-coated with anti-IGF-I receptor beta chain C-terminus monoclonal antibody TC123 and was incubated with the lysate samples for 5 h at ambient temperature to capture IGF-I receptor. The wells containing the captured IGF-I receptor were then washed and incubated with biotinylated anti-phosphotyrosine antibody (PY20; 0.25 μg/mL; BD Transduction Laboratories) for 30 min, followed by washes and incubation with streptavidin-horseradish peroxidase conjugate (0.8 μg/mL) for 30 min. The wells were washed and detected with ABTS/H 2 O 2  substrate. Use of a control anti-B4 antibody showed no inhibition of the IGF-I stimulated autophosphorylation of IGF-I receptor. In contrast, a complete inhibition of the IGF-I stimulated autophosphorylation of IGF-I receptor was obtained upon treatment with EM164 antibody ( FIG. 4 ). 
         [0113]    To demonstrate inhibition of phosphorylation of insulin receptor substrate-1 (IRS-1), an ELISA using immobilized anti-IRS-1 antibody to capture IRS-1 from lysates was used, followed by measurement of the associated p85 subunit of phosphatidylinositol-3-kinase (PI-3-kinase) that binds to the phosphorylated IRS-1 (Jackson, J. G. et al., 1998,  J. Biol. Chem.,  273, 9994-10003). In  FIG. 5 , MCF-7 cells were treated with 5 μg/mL antibody (EM164 or IR3) in serum-free medium for 2 h, followed by stimulation with 50 ng/mL IGF-I for 10 min at 37° C. Anti-IRS-1 antibody (rabbit polyclonal; Upstate Biotechnology) was indirectly captured by incubation with coated goat-anti-rabbit-IgG antibody on an ELISA plate, which was then used to capture IRS-1 from the cell lysate samples by overnight incubation at 4° C. The wells were then incubated with mouse monoclonal anti-p85-PI-3-kinase antibody (Upstate Biotechnology) for 4 h, followed by treatment with goat-anti-mouse-IgG-antibody-HRP conjugate for 30 min. The wells were then washed and detected using ABTS/H 2 O 2  substrate ( FIG. 5 ). As shown in  FIG. 5 , EM164 antibody was more effective at inhibiting the IGF-I-stimulated IRS-1 phosphorylation than was IR3 antibody, and EM164 antibody did not show any agonistic activity on IRS-1 phosphorylation when incubated with cells in the absence of IGF-I. 
         [0114]    The activation of other downstream effectors, such as Akt and Erk1/2, was also inhibited in a dose-dependent manner by EM164 antibody in SaOS-2 cells ( FIG. 6 ) and in MCF-7 cells, as was shown using Western blots of lysates and phosphorylation-specific antibodies (rabbit polyclonal anti-phospho-Ser 473  Akt polyclonal and anti-phospho-ERK1/2 antibodies; Cell Signaling Technology). A pan-ERK antibody demonstrated equal protein loads in all lanes ( FIG. 6 ). Treatment of SaOS-2 cells with EM164 antibody did not inhibit the EGF-stimulated phosphorylation of Erk1/2, thus demonstrating the specificity of inhibition of IGF-I receptor signaling pathway by EM164 antibody. 
       E. Inhibition of IGF-I-, IGF-II- and Serum-Stimulated Growth and Survival of Human Tumor Cells by EM164 Antibody 
       [0115]    Several human tumor cell lines were 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 using an MTT assay after 2-4 days. Approximately 1500 cells were plated in a 96-well plate in regular medium with serum, which was replaced with serum-free medium the following day (either serum-free RPMI medium supplemented with transferrin and BSA, or phenol-red free medium as specified by Dufourny, B. et al., 1997 , J. Biol. Chem.,  272, 31163-31171). After one day of growth in serum-free medium, the cells were incubated with about 75 μL of 10 μg/mL antibody for 2-3 h, followed by the addition of 25 μL of IGF-I (or IGF-II or serum) solution to obtain a final concentration of 10 ng/mL IGF-I, or 20 ng/mL IGF-II, or 0.04-10% serum. The cells were then allowed to grow for another 2-3 days. A solution of MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 25 μL of a 5 mg/mL solution in PBS) was then added and the cells were returned to the incubator for 2-3 h. The medium was then removed and replaced by 100 μL DMSO, mixed, and the absorbance of the plate was measured at 545 nm. Several human tumor cell lines showed a growth and survival response upon addition of IGF-I or IGF-II or serum that was significantly inhibited by EM164 antibody (Table 1). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 INHIBITION OF IGF-I-STIMULATED GROWTH AND SURVIVAL OF 
               
               
                 TUMOR CELLS BY EM164 ANTIBODY 
               
             
          
           
               
                   
                   
                 % Inhibition by 
                   
               
               
                   
                 Fold growth response 
                 EM164 antibody 
                 Inhibition by 
               
               
                   
                 to IGF-I (MTT assay: 
                 of IGF-I- 
                 EM164 antibody of 
               
               
                   
                 ratio for IGF-I treated 
                 stimulated growth 
                 Growth/survival of 
               
               
                   
                 vs untreated cells in 
                 in serum-free 
                 cells in 1.25-10% 
               
               
                 Tumor Cell Type 
                 serum-free medium) a   
                 medium 
                 serum b   
               
               
                   
               
               
                 MCF-7 (breast) 
                 1.7-2.8 
                 100%  
                 85% 
               
               
                 HT-3 (cervical) 
                 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 (rhabdomyocarcoma) 
                 1.7 
                 85-100% 
                 Yes 
               
               
                 SaOS-2 (osteosarcoma) 
                 2.5 
                 100%  
                 Yes 
               
               
                 A 431 (epidermoid) 
                 2.2 
                 85% 
                 Yes 
               
               
                 SK-N-SH (neuroblastoma) 
                 2 
                 85% 
                 30-50% 
               
               
                   
               
               
                   a MTT assay of 3- to 4-day growth/survival of cells in response to 10 ng/mL IGF-I in serum-free medium containing 5-10 μg/mL EM164 antibody. 
               
               
                   b inhibition of growth of cells in 1.25-10% serum in the presence of 5-10 μg/mL EM164 antibody by MTT assay or colony formation assay based on comparison with the control (with serum but without antibody); the extent of inhibition was quantitatively measured for MCF-7, NCI-H838 and SK-N-SH cells based on controls (without serum but with antibody, and with serum but without antibody) to account for autocrine/paracrine IGF-stimulation by cells. 
               
               
                 ND indicates no data or poor data due to staining difficulties. 
               
             
          
         
       
     
         [0116]    The EM164 antibody strongly inhibited IGF-I- or serum-stimulated growth and survival of breast cancer MCF-7 cells ( FIGS. 7 and 8 ). In a separate experiment, the EM164 antibody strongly inhibited IGF-II-stimulated growth and survival of MCF-7 cells. Previous reports using commercially available antibodies such as IR3 antibody showed only weak inhibition of serum-stimulated growth and survival of MCF-7 cells, as confirmed in  FIG. 7  for the IR3 and 1H7 antibodies (Cullen, K. J. et al., 1990,  Cancer Res.,  50, 48-53). In contrast, EM164 antibody was a potent inhibitor of the serum- or IGF-stimulated growth of MCF-7 cells. As shown in  FIG. 8 , EM164 antibody was equally effective in inhibiting the growth and survival of MCF-7 cells over a wide range of serum concentrations (0.04-10% serum). 
         [0117]    The growth inhibition of MCF-7 cells by EM164 antibody was measured by counting cells. Thus, in a 12-well plate, about 7500 cells were plated in RPMI medium with 10% FBS, in the presence or absence of 10 μg/mL EM164 antibody. After 5 days of growth, the cell count for the untreated control sample was 20.5×10 4  cells, in contrast to a cell count of only 1.7×10 4  cells for the EM164 antibody-treated sample. Treatment with the EM164 antibody inhibited the growth of MCF-7 cells by about 12-fold in 5 days. This inhibition by EM164 antibody was significantly greater than was a reported 2.5-fold inhibition using IR3 antibody in a 6-day assay for MCF-7 cells (Rohlik, Q. T. et al., 1987 , Biochem. Biophys. Res. Commun.,  149, 276-281). 
         [0118]    The IGF-I- and serum-stimulated growth and survival of a non-small cell lung cancer line NCI-H838 were also strongly inhibited by EM164 antibody, compared to a control anti-B4 antibody ( FIG. 9 ). Treatment with EM164 antibody in serum-free medium produced a smaller signal than the untreated sample for both NCI-H838 and MCF-7 cells, presumably because EM164 antibody also inhibited the autocrine and paracrine IGF-I and IGF-II stimulation of these cells ( FIGS. 7 and 9 ). The colony size of HT29 colon cancer cells was also greatly reduced upon treatment with EM164 antibody. 
         [0119]    EM164 antibody is therefore unique among all known anti-IGF-I receptor antibodies in its effectiveness to inhibit the serum-stimulated growth of tumor cells such as MCF-7 cells and NCI-H838 cells by greater than 80%. 
         [0000]    F. Synergistic Inhibition by EM164 Antibody of Growth and Survival of Human Tumor Cells in Combinations with Other Cytotoxic and Cytostatic Agents 
         [0120]    The combined administration of EM164 antibody with taxol was significantly more inhibitory to the growth and survival of non-small cell lung cancer Calu6 cells than was taxol alone. Similarly, the combination of EM164 antibody with camptothecin was significantly more inhibitory than camptothecin alone toward the growth and survival of colon cancer HT29 cells. Because EM164 antibody alone was not expected to be as toxic to cells as organic chemotoxic drugs, the synergism between the predominantly cytostatic effect of EM164 antibody and the cytotoxic effect of the chemotoxic drug may be highly efficacious in combination cancer therapies in clinical settings. 
         [0121]    The combined effect of EM164 antibody with an anti-EGF receptor antibody (KS77) was significantly more inhibitory than either EM164 antibody or KS77 antibody alone on the growth and survival of several tumor cell lines such as HT-3 cells, RD cells, MCF-7 cells, and A431 cells. Therefore, the synergistic effect of combining neutralizing antibodies for two growth factor receptors such as IGF-I receptor and EGF receptor may also be useful in clinical cancer treatment. 
         [0122]    Conjugates of EM164 antibody with cytotoxic drugs are also valuable in targeted delivery of the cytotoxic drugs to the tumors overexpressing IGF-I receptor. Conjugates of EM164 antibody with radiolabels or other labels can be used in the treatment and imaging of tumors that overexpress IGF-I receptor. 
         [0000]    G. EM164 Antibody Treatment Alone, and in Combination with Taxol, on Mice with Human Tumor Calu-6 Xenograft 
         [0123]    Human non-small cell lung cancer Calu-6 xenografts were established in mice by subcutaneous injections of 1×10 7  Calu-6 cells. As shown in  FIG. 10 , these mice containing established 100 mm 3  Calu-6 xenografts were treated with EM164 antibody alone (6 injections of 0.8 mg/mouse, i. v., two per week) or with taxol alone (five injections of taxol, i.p. every two days; 15 mg/kg), or with a combination of taxol and EM164 antibody treatments, or PBS alone (200 μL/mouse, 6 injections, two per week, i.v.) using five mice per treatment group. The growth of tumors was significantly slowed by EM164 antibody treatment compared to a PBS control. No toxicity of EM164 antibody was observed, based on measurements of the weights of the mice. Although taxol treatment alone was effective until day 14, the tumor then started to grow back. However, the growth of the tumor was delayed significantly in the group that was treated by a combination of taxol and EM164 antibody, compared to the group that was treated with taxol alone. 
       H. Cloning and Sequencing of the Light and Heavy Chains of EM164 Antibody 
       [0124]    Total RNA was purified from EM164 hybridoma cells. Reverse transcriptase reactions were performed using 4-5 μg total RNA and either oligo dT or random hexamer primers. 
         [0125]    PCR reactions were performed using a RACE method described in Co et al. (J. Immunol., 148, 1149-1154 (1992)) and using degenerate primers as described in Wang et al., (J. Immunol. Methods, 233, 167-177 (2000)). The RACE PCR method required an intermediate step to add a poly G tail on the 3′ ends of the first strand cDNAs. RT reactions were purified with Qianeasy (Qiagen) columns and eluted in 50 μl 1×NEB buffer 4. A dG tailing reaction was performed on the eluate with 0.25 mM CoCl 2 , 1 mM dGTP, and 5 units terminal transferase (NEB), in 1×NEB buffer 4. The mixture was incubated at 37° C. for 30 minutes and then 1/5 of the reaction (10 μl) was added directly to a PCR reaction to serve as the template DNA. 
         [0126]    The RACE and degenerate PCR reactions were identical except for differences in primers and template. The terminal transferase reaction was used directly for the RACE PCR template, while the RT reaction mix was used directly for degenerate PCR reactions. 
         [0127]    In both RACE and degenerate PCR reactions the same 3′ light chain primer: 
         [0000]                                                        HindKL-                (SEQ ID NO:14)                    tatagagctcaagcttggatggtgggaagatggatacagttggtgc                
and 3′ heavy chain primer:
 
         [0000]                                      (SEQ ID NO: 15)                    Bg12IgG1-ggaagatctatagacagatgggggtgtcgttttggc                
were used.
 
         [0128]    In the RACE PCR, one poly C 5′ primer was used for both the heavy and light chain: 
         [0000]                                      (SEQ ID NO:16)                    EcoPolyC-TATATCTAGAATTCCCCCCCCCCCCCCCCC,                
while the degenerate 5′ end PCR primers were:
 
         [0000]                                      (SEQ ID NO:17)                    Sac1MK-GGGAGCTCGAYATTGTGMTSACMCARWCTMCA                
for the light chain, and an equal mix of:
 
         [0000]                                                              (SEQ ID NO:18)                    EcoR1MH1-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC               and                        (SEQ ID NO:19)                    EcoR1MH2-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG                
for the heavy chain.
 
         [0129]    In the above primer sequences, mixed bases are 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. 
         [0130]    The PCR reactions were performed using the following program: 1) 94° C. 3 min, 2) 94° C. 15 sec, 3) 45° C. 1 min, 4) 72° C. 2 min, 5) cycle back to step #2 29 times, 6) finish with a final extension step at 72° C. for 10 min. 
         [0131]    The PCR products were cloned into pBluescript II SK+ (Stratagene) using restriction enzymes created by the PCR primers. 
         [0132]    Several individual light and heavy chain clones were sequenced by conventional means to identify and avoid possible polymerase generated sequence errors ( FIGS. 12 and 13 ). Using Chothia canonical classification definitions, the three light chain and heavy chain CDRs were identified ( FIGS. 12-14 ). 
         [0133]    A search of the NCBI IgBlast database indicated that the anti-IGF-I receptor antibody light chain variable region probably derived from the mouse IgVk Cr1 germline gene while the heavy chain variable region probably derived from the IgVh J558.c germline gene ( FIG. 15 ). 
         [0134]    Protein sequencing of murine EM164 antibody was performed to confirm the sequences shown in  FIGS. 12 and 13 . The heavy and light chain protein bands of purified EM164 antibody were transferred to a PVDF membrane from a gel (SDS-PAGE, reducing conditions), excised from the PVDF membrane and analyzed by protein sequencing. The N-terminal sequence of the light chain was determined by Edman sequencing to be: DVLMTQTPLS (SEQ ID NO:20), which matches the N-terminal sequence of the cloned light chain gene obtained from the EM164 hybridoma. 
         [0135]    The N-terminus of the heavy chain was found to be blocked for Edman protein sequencing. A tryptic digest peptide fragment of the heavy chain of mass 1129.5 (M+H + , monoisotopic) was fragmented via post-source decay (PSD) and its sequence was determined to be GRPDYYGSSK (SEQ ID NO:21). Another tryptic digest peptide fragment of the heavy chain of mass 2664.2 (M+H + , monoisotopic) was also fragmented via post-source decay (PSD) and its sequence was identified as: SSSTAYMQLSSLTSEDSAVYYFAR (SEQ ID NO:22). Both of these sequences match perfectly those of CDR3 and framework 3 (FR3) of the cloned heavy chain gene obtained from the EM164 hybridoma. 
       I. Recombinant Expression of EM164 Antibody 
       [0136]    The light and heavy chain paired sequences were cloned into a single mammalian expression vector ( FIG. 16 ). The PCR primers for the human variable sequences created restriction sites that allowed the human signal sequence to be attached while in the pBluescriptII cloning vector, and the variable sequences were cloned into the mammalian expression plasmid using EcoRI and BsiWI or HindIII and ApaI sites for the light chain or heavy chain, respectively ( FIG. 16 ). The light chain variable sequences were cloned in-frame onto the human IgK constant region and the heavy chain variable sequences were cloned into the human Iggamma1 constant region sequence. In the final expression plasmids, human CMV promoters drove the expression of both the light and heavy chain cDNA sequences. Expression and purification of the recombinant mouse EM164 antibody proceeded according to methods that are well-known in the art. 
       Example 2 
     Humanized Versions of EM164 Antibody 
       [0137]    Resurfacing of the EM164 antibody to provide humanized versions suitable as therapeutic or diagnostic agents generally proceeds according to the principles and methods disclosed in U.S. Pat. No. 5,639,641, and as follows. 
       A. Surface Prediction 
       [0138]    The solvent accessibility of the variable region residues for a set of antibodies with solved structures was used to predict the surface residues for the murine anti-IGF-I receptor antibody (EM164) variable region. The amino acid solvent accessibility for a set of 127 unique antibody structure files (Table 2) were calculated with the MC software package (Pedersen et al., 1994,  J. Mol. Biol.,  235, 959-973). The ten most similar light chain and heavy chain amino acid sequences from this set of 127 structures were determined by sequence alignment. The average solvent accessibility for each variable region residue was calculated, and positions with greater than a 30% average accessibility were considered to be surface residues. Positions with average accessibilities of between 25% and 35% were further examined by calculating the individual residue accessibility for only those structures with two identical flanking residues. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 127 antibody structures from the Brookhaven database used to 
               
               
                 predict the surface of anti-IGF-I-receptor antibody (EM164) 
               
               
                 127 Brookhaven structure files used for surface predictions 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 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 
               
               
                 1nsn 
                 1opg 
                 1osp 
                 1aj7 
                 1ay1 
                 1clz 
                 1plg 
                 1psk 
                 1rmf 
                 1sbs 
               
               
                 1ncd 
                 1nfd 
                 1ngp 
                 1acy 
                 1afv 
                 1cbv 
                 1nld 
                 1nma 
                 1nmb 
                 1nqb 
               
               
                 1mcp 
                 1mfb 
                 1mim 
                 15c8 
                 1a5f 
                 1axs 
                 1mlb 
                 1mpa 
                 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. Molecular Modeling: 
       [0139]    A molecular model of murine EM164 was generated using the Oxford Molecular software package AbM. The antibody framework was built from structure files for the antibodies with the most similar amino acid sequences, which were 2jel for the light chain and 1nqb for the heavy chain. The non-canonical CDRs were built by searching a C-α structure database containing non-redundant solved structures. Residues that lie within 5 Å of a CDR were determined. 
       C. Human Ab Selection 
       [0140]    The surface positions of murine EM164 were compared to the corresponding positions in human antibody sequences in the Kabat database (Johnson, G. and Wu, T. T. (2001)  Nucleic Acids Research,  29: 205-206). The antibody database management software SR (Searle 1998) was used to extract and align the antibody surface residues from natural heavy and light chain human antibody pairs. The human antibody surface with the most identical surface residues, with special consideration given to positions that come within 5 Å of a CDR, was chosen to replace the murine anti-IGF-I receptor antibody surface residues. 
       D. PCR Mutagenesis 
       [0141]    PCR mutagenesis was performed on the murine EM164 cDNA clone (above) to build the resurfaced, human EM164 (herein huEM164). Primer sets were designed to make the 8 amino acid changes required for all tested versions of huEM164, and additional primers were designed to alternatively make the two 5 Å residue changes (Table 3). PCR reactions were performed with the following program: 1) 94° C. 1 min, 2) 94° C. 15 sec, 3) 55° C. 1 min, 4) 72° C. 1 min, 5) cycle back to step #2 29 times, 6) finish with a final extension step at 72° C. for 4 min. The PCR products were digested with their corresponding restriction enzymes and were cloned into the pBluescript cloning vectors as described above. Clones were sequenced to confirm the desired amino acid changes. 
         [0000]    
       
         
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 PCR primers used to build 4 humanized EM164 
                   
               
               
                 antibodies 
               
             
          
           
               
                   
                   
                 SEQ 
                   
               
               
                   
                   
                 ID 
               
               
                 Primer 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 Em164hcvv 
                 CAGGTGTACACTCCCAGGTCCAACTGGTGCAGTC 
                 23 
                   
               
               
                   
                 TGGGGCTGAAGTGGTGAAGCCTG 
               
               
                   
               
               
                 Em164hqqggol1 
                 CAATCAGAAGTTCCAGGGGAAGGCCACAC 
                 24 
               
               
                   
               
               
                 Em164hcqqgol2 
                 CCTTCCCCTGGAACTTCTGATTGTAGTTAGTACG 
                 25 
               
               
                   
               
               
                 Em164lcv3 
                 CAGGTGTACACTCCGATGTTGTGATGACCCAAAC 
                 26 
               
               
                   
                 TCC 
               
               
                   
               
               
                 Em164lc13 
                 CAGGTGTACACTCCGATGTTTTGATGACCCAAAC 
                 27 
               
               
                 TCC 
               
               
                   
               
               
                 Em164lcp18 
                 GACTAGATCTGCAAGAGATGGAGGCTGGATCTCC 
                 28 
               
               
                   
                 AAGAC 
               
               
                   
               
               
                 Em164lcbg12 
                 TTGCAGATCTAGTCAGAGCATAGTACATAGTAAT 
                 29 
               
               
                   
                 G 
               
               
                   
               
               
                 Em164r45 
                 GAATGGTACCTGCAGAAACCAGGCCAGTCTCCAA 
                 30 
               
               
                   
                 GGCTCCTGATCTAC 
               
               
                   
               
               
                 Em164a67ol1 
                 GTGGCAGTGGAGCAGGGACAGATTTCAC 
                 31 
               
               
                   
               
               
                 Em164a67ol2 
                 GAAATCTGTCCCTGCTCCACTGCCACTG 
                 32 
               
               
                   
               
             
          
         
       
     
       E. Variable Region Surface Residues 
       [0142]    The antibody resurfacing techniques described by Pedersen et al. ( J. Mol. Biol.,  235, 959-973, 1994) and Roguska et al. (Protein Eng., 9, 895-904, 1996) begin by predicting the surface residues of the murine antibody variable sequences. A surface residue is defined as an amino acid that has at least 30% of its total surface area accessible to a water molecule. 
         [0143]    The 10 most homologous antibodies in the set of 127 antibody structure files were identified ( FIGS. 17 and 18 ). The solvent accessibility for each Kabat position was averaged for these aligned sequences and the distribution of the relative accessibilities for each residue were as shown in  FIG. 19 . Both the light and heavy chain have 26 residues with average relative accessibilities of at least 30% ( FIG. 19 ): these residues were therefore the predicted surface residues for EM164. Several residues had average accessibilities of between 25% and 35%, and these were further examined by averaging only the antibodies with two identical residues flanking either side of the residue (Tables 4 and 5). After this additional analysis, the original set of surface residues that was identified above remained unchanged. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Surface residues and average accessibility (ave. acc.) for the 
               
               
                 light and heavy chain variable sequences of EM164 antibody 
               
               
                 EM164 Surface Residues 
               
             
          
           
               
                 Light Chain 
                 Heavy Chain 
               
             
          
           
               
                 EM164 
                 Kabat # 
                 Ave. Acc. 
                 EM164 
                 Kabat # 
                 Ave. Acc. 
               
               
                   
               
               
                 D 
                  1 
                 45.89 
                 Q 
                  1 
                 58.19 
               
               
                 L 
                  3 
                 41.53 
                 Q 
                  3 
                 34.08 
               
               
                 T 
                  7 
                 31.40 
                 Q 
                  5 
                 34.36 
               
               
                 L 
                  9 
                 50.08 
                 A 
                  9 
                 38.01 
               
               
                 L 
                 15 
                 35.45 
                 L 
                 11 
                 47.72 
               
               
                 Q 
                 18 
                 39.79 
                 K 
                 13 
                 46.51 
               
               
                 R 
                 24 
                 34.36 
                 P 
                 14 
                 31.49 
               
               
                 S 
                 26 
                 32.63 
                 G 
                 15 
                 31.42 
               
               
                 Q 
                 27 
                 34.35 
                 K 
                 19 
                 34.41 
               
               
                 N 
                 28 
                 36.38 
                 K 
                 23 
                 31.23 
               
               
                 P 
                 40 
                 43.05 
                 T 
                 28 
                 36.24 
               
               
                 G 
                 41 
                 46.56 
                 P 
                 41 
                 44.01 
               
               
                 Q 
                 42 
                 34.92 
                 G 
                 42 
                 42.62 
               
               
                 K 
                 45 
                 30.58 
                 Q 
                 43 
                 46.85 
               
               
                 S 
                 52 
                 30.40 
                 E 
                 61 
                 46.68 
               
               
                 S 
                 56 
                 41.46 
                 K 
                 62 
                 44.87 
               
               
                 G 
                 57 
                 42.41 
                 K 
                 64 
                 38.92 
               
               
                 D 
                 60 
                 45.96 
                 R 
                 65 
                 40.06 
               
               
                 S 
                 67 
                 38.20 
                 K 
                 73 
                 35.92 
               
               
                 R 
                 77 
                 42.61 
                 S 
                 74 
                 48.91 
               
               
                 E 
                 81 
                 38.46 
                 S 
                   82B 
                 32.72 
               
               
                 V 
                   95E 
                 34.83 
                 S 
                 84 
                 35.21 
               
               
                 K 
                 103  
                 31.10 
                 E 
                 85 
                 39.62 
               
               
                 K 
                 107  
                 36.94 
                 D 
                 98 
                 36.00 
               
               
                 R 
                 108  
                 60.13 
                 A 
                 106  
                 37.65 
               
               
                 A 
                 109  
                 53.65 
                 S 
                 113  
                 43.42 
               
               
                   
               
             
          
         
       
     
         [0000]                                                        TABLE 5                   Residues which had average accessibilities between 25% and 35%       were further analyzed by averaging a subset of antibodies that       had two identical residues flanking either side of the residue in       question. These borderline surface positions and their new average       accessibilities are given. The NA&#39;s refer to residues with no       identical flanking residues in the 10 most similar antibodies.       Borderline Surface Residues            Light Chain   Heavy Chain            EM164   Kabat #   Ave. Acc.   EM164   Kabat #   Ave. Acc.               T    5   28.68   Q    3   31.62       T    7   30.24   Q    5   36.07       P   12   26.59   P   14   29.88       G   16   25.20   G   15   30.87       D   17   25.73   S   17   25.64       S   20   25.37   K   19   35.06       R   24   36.73   K   23   31.48       S   26   31.00   G   26   30.53       Q   27   32.29   S   31   27.12       S     27A   29.78   R   56   NA       V     27C   29.05   T   68   27.71       V   29   NA   T   70   24.65       Q   42   34.92   S   75   18.80       K   45   32.24   S     82B   32.87       S   52   30.02   P   97   NA       R   54   29.50   Y   99   NA       D   70   26.03   V   103    NA       R   74   NA   T   111    25.95       E   79   26.64       A   80   29.61       V     95E   42.12       G   100    29.82       K   103    31.10       E   105    25.78                    
F. Molecular Modeling to Determine which Residues Fall within 5 Å of a CDR
 
         [0144]    The molecular model above, generated with the AbM software package, was analyzed to determine which EM164 surface residues were within 5 Å of a CDR. In order to resurface the murine EM164 antibody, all surface residues outside of a CDR should be changed to the human counterpart, but residues within 5 Å of a CDR are treated with special care because they may also contribute to antigen specificity. Therefore, these latter residues must be identified and carefully considered throughout the humanization process. The CDR definitions used for resurfacing combine the AbM definition for heavy chain CDR2 and Kabat definitions for the remaining 5 CDRs ( FIG. 14 ). Table 6 shows the residues that were within 5 Å of any CDR residue in either the light or heavy chain sequence of the EM164 model. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 EM164 antibody framework surface residues within 5Å of a CDR 
               
               
                 EM164 Surface Residues 
               
               
                 within 5 Å of a CDR 
               
             
          
           
               
                   
                 Light chain 
                 Heavy chain 
               
               
                   
                   
               
               
                   
                 D1 
                 T28 
               
               
                   
                 L3 
                 K73 
               
               
                   
                 T7 
                 S74 
               
               
                   
                 P40 
               
               
                   
                 Q42 
               
               
                   
                 K45 
               
               
                   
                 G57 
               
               
                   
                 D60 
               
               
                   
                 E81 
               
               
                   
                   
               
             
          
         
       
     
       G. Identification of the Most Homologous Human Surfaces 
       [0145]    Candidate human antibody surfaces for resurfacing EM164 were identified within the Kabat antibody sequence database using SR software, which provided for the searching of only specified residue positions against the antibody database. To preserve the natural pairings, surface residues of both the light and heavy chains were compared together. The most homologous human surfaces from the Kabat database were aligned in rank order of sequence identity. The top 5 surfaces are given in Table 7. These surfaces were then compared to identify which of them would require the least changes within 5 Å of a CDR. The Leukemic B-cell antibody, CLL 1.69, required the least number of surface residue changes (10 in total) and only two of these residues were within 5 Å of a CDR. 
         [0146]    The full length variable region sequence for EM164 was also aligned against the Kabat human antibody database and CLL 1.69 was again identified as the most similar human variable region sequence. Together, these sequence comparisons identified the human Leukemic B-cell antibody CLL 1.69 as the preferred choice as a human surface for EM164. 
         [0000]    
       
         
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 The top 5 human sequences extracted from the Kabat database 
                   
               
               
                 Alignments were generated by SR (Pedersen 1993). The EM164 
               
               
                 surface residues that come within 5Å of a CDR are 
               
               
                 underlined. 
               
               
                 5 Most Homologous Human Antibody Surfaces 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 SEQ 
                   
               
               
                   
                   
                 ID 
               
               
                 Antibody 
                 Light Chain 
                 NO: 
               
               
                   
               
               
                 MuEM164 
                   D L T  L L Q  P  G  Q K G D  S R  E  K K R A 
                 33 
               
               
                 CLL1.69 
                 D V T L L P P G Q R G D A R E K K R - 
                 34 
               
               
                 MSL5 
                 D Q S L I P P G Q K G D S R D K K R A 
                 35 
               
               
                 CDP571 
                 D M S S V R P G Q K G S S S D K K R - 
                 36 
               
               
                 LC3aPB 
                 E V S G P R P G Q R G D S R E K K R - 
                 37 
               
               
                 SSbPB 
                 E V S G P R P G Q R G D S R E K K R - 
                 38 
               
               
                   
               
               
                 Antibody 
                 Heavy Chain 
               
               
                   
               
               
                 MuEM164 
                 Q Q Q A L K P G K K  T  P G Q E K K R  K S  S S E A S 
                 39 
               
               
                 CLL1.69 
                 Q Q V A V K P G K K T P G Q Q K Q G K S S S E Q S 
                 40 
               
               
                 MSL5 
                 Q Q Q P L K P G K K T P G K D D K G T S N N E Q S 
                 41 
               
               
                 CDP571 
                 Q Q V A V K P G K K T P G Q Q K K G K S S S E Q S 
                 42 
               
               
                 LC3aPB 
                 - Q V A V K P G K K T P G Q Q K Q G K S S S E Q S 
                 43 
               
               
                 SSbPB 
                 - Q V A V K P G K K T P G Q Q K Q G E S S S E Q S 
                 44 
               
               
                   
               
             
          
         
       
     
       H. Construction of Humanized EM164 Genes 
       [0147]    The ten surface residue changes for EM164 (Table 7) were made using PCR mutagenesis techniques as described above. Because eight of the surface residues for CLL 1.69 were not within 5 Å of a CDR, these residues were changed from murine to human in all versions of humanized EM164 (Tables 8 and 9). The two light chain surface residues that were within 5 Å of a CDR (Kabat positions 3 and 45) were either changed to human or were retained as murine. Together, these options generate the four humanized versions of EM164 that were constructed ( FIGS. 22 and 23 ). 
         [0148]    Of the four humanized versions, version 1.0 has all 10 human surface residues. The most conservative version with respect to changes in the vicinity of the CDR is version 1.1, which retained both of the murine surface residues that were within 5 Å of a CDR. All four humanized EM164 antibody genes were cloned into an antibody expression plasmid ( FIG. 16 ) for use in transient and stable transfections. 
         [0000]                                                                                                    TABLE 8               Residue changes for versions 1.0-1.3 of humanized EM164 antibody                   Changes in all versions                    Light Chain:   muQ18 to huP18;   muS67 to huA67               Heavy Chain:   muQ5 to huV5;   muL11 to huV11;   muE61 to huQ61;   muK64 to huQ64;           muR65 to huG65;   muA106 to huQ106                    huEM164 changes                    Light Chain aa3       Light Chain aa45   Total 5A                Mu   hu   mu   hu   Mouse Res               v1.0       V       R   0       V1.1   L       K       2       v1.2   L           R   1       V1.3       V   K       1                    
I. Comparison of the Affinities of Humanized EM164 Antibody Versions with Murine EM164 Antibody for Binding to Full-Length IGF-I Receptor and to Truncated IGF-I Receptor Alpha Chain
 
         [0149]    The affinities of the humanized EM164 antibody versions 1.0-1.3 were compared to those of murine EM164 antibody through binding competition assays using biotinylated full-length human IGF-I receptor or myc-epitope tagged truncated IGF-I receptor alpha chain, as described above. Humanized EM164 antibody samples were obtained by transient transfection of the appropriate expression vectors in human embryonic kidney 293T cells, and antibody concentrations were determined by ELISA using purified humanized antibody standards. For ELISA binding competition measurements, mixtures of humanized antibody samples and various concentrations of murine EM164 antibody were incubated with indirectly captured biotinylated full-length IGF-I receptor or myc-epitope tagged truncated IGF-I receptor alpha chain. After equilibration, the bound humanized antibody was detected using a goat-anti-human-Fab′ 2 -antibody-horseradish peroxidase conjugate. Plots of ([bound murine Ab]/[bound humanized Ab]) vs ([murine Ab]/[humanized Ab]), which theoretically yield a straight line with slope=(K d humanized Ab /K d murine Ab ), were used to determine the relative affinities of the humanized and murine antibodies. 
         [0150]    An exemplary competition assay is shown in  FIG. 11 . An Immulon-2HB ELISA plate was coated with 100 μL of 5 μg/mL streptavidin per well in carbonate buffer at ambient temperature for 7 h. The streptavidin-coated wells were blocked with 200 μL of blocking buffer (10 mg/mL BSA in TBS-T buffer) for 1 h, washed with TBS-T buffer and incubated with biotinylated IGF-I receptor (5 ng per well) overnight at 4° C. The wells containing indirectly captured biotinylated IGF-I receptor were then washed and incubated with mixtures of humanized EM164 antibody (15.5 ng) and murine antibody (0 ng, or 16.35 ng, or 32.7 ng, or 65.4 ng, or 163.5 ng) in 100 μL blocking buffer for 2 h at ambient temperature and were then incubated overnight at 4° C. The wells were then washed with TBS-T buffer and incubated with goat-anti-human-Fab′ 2 -antibody-horseradish peroxidase conjugate for 1 h (100 μL; 1 μg/mL in blocking buffer), followed by washes and detection using ABTS/H 2 O 2  substrate at 405 nm. The plot of ([bound murine Ab]/[bound humanized Ab]) vs ([murine Ab]/[humanized Ab]) yielded a straight line (r 2 =0.996) with slope (=K d humanized Ab /K d murine Ab ) of 0.52. The humanized antibody version 1.0 therefore bound to IGF-I receptor more tightly than did murine EM164 antibody. Similar values for the gradient, ranging from about 0.5 to 0.8, were obtained for competitions of versions 1.0, 1.1, 1.2 and 1.3 of humanized EM164 antibodies with murine EM164 antibody for binding to full-length IGF-I receptor or to truncated IGF-I receptor alpha chain, which indicated that all of the humanized versions of EM164 antibody had similar affinities, which were all better than that of the parent murine EM164 antibody. A chimeric version of EM164 antibody with 92F→C mutation in heavy chain showed a slope of about 3 in a similar binding competition with murine EM164 antibody, which indicated that the 92F→C mutant of EM164 had a 3-fold lower affinity than did murine EM164 antibody for binding to IGF-I receptor. The humanized EM164 v1.0 antibody showed a similar inhibition of IGF-I-stimulated growth and survival of MCF-7 cells as did the murine EM164 antibody ( FIG. 24 ). The inhibition of serum-stimulated growth and survival of MCF-7 cells by humanized EM164 v1.0 antibody was similar to the inhibition by murine EM164 antibody. 
         [0000]                                  TABLE 9                   The Kabat numbering system is used for the light chain and heavy chain       variable region polypeptides of the different versions of the EM164 Ab.       The amino acid residues are grouped into Framework (FR) and       Complementarity Determining Regions (CDR) according to positio       in the polypeptide chain.       Taken from Kabat et al. Sequences of Proteins of Immunological Interest,       Fifth Edition, 1991, NIH Publication No. 91-3242            Segment   Light Chain   Heavy Chain               FR1    1-23 (with an occasional residue    1-30 (with an           at 0, and a deletion at 10 in   occasional residue at 0)           V λ  chains)       CDR1   24-34 (with possible insertions    31-35 (with possible           numbered as 27A, B, C, D, E, F)   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    95-102 (with possible           numbered as 95A, B, C, D, E, F)   insertions numbered as               100A, B, C, D, E, F, G,               H, I, J, K)       FR4   98-107 (with a possible insertion   103-113           numbered as 106A)                    
J. Process of Providing Improved Anti-IGF-I-Receptor Antibodies Starting from the Murine and Humanized Antibody Sequences Described Herein:
 
         [0151]    The amino acid and nucleic acid sequences of the anti-IGF-I receptor antibody EM164 and its humanized variants were used to develop other antibodies that have improved properties and that are also within the scope of the present invention. Such improved properties include increased affinity for the IGF-I receptor. Several studies have surveyed the effects of introducing one or more amino acid changes at various positions in the sequence of an antibody, based on the knowledge of the primary antibody sequence, on its properties such as binding and level of expression (Yang, W. P. et al., 1995,  J. Mol. Biol.,  254, 392-403; Rader, C. et al., 1998 , Proc. Natl. Acad. Sci. USA,  95, 8910-8915; Vaughan, T. J. et al., 1998,  Nature Biotechnology,  16, 535-539). 
         [0152]    In these studies, variants of the primary antibody have been generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2, CDR3, or framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of  E. coli  (Vaughan, T. J. et al., 1998,  Nature Biotechnology,  16, 535-539; Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “ Phage Display of Peptides and Proteins ”, Eds. Kay, B. K. et al., Academic Press). These methods of changing the sequence of the primary antibody have resulted, through the use of standard screening techniques, in improved affinities of such secondary antibodies (Gram, H. et al., 1992 , Proc. Natl. Acad. Sci. USA,  89, 3576-3580; Boder, E. T. 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, M. K. et al., 2002,  J. Biol. Chem.,  277, 16365-16370; Furukawa, K. et al., 2001,  J. Biol. Chem.,  276, 27622-27628). 
         [0153]    By a similar directed strategy of changing one or more amino acid residues of the antibody, the antibody sequences described in this invention can be used to develop anti-IGF-I receptor antibodies with improved functions, such as antibodies having suitable groups such as free amino groups or thiols at convenient attachment points for covalent modification for use, for example, in the attachment of therapeutic agents. 
       K. Alternative Expression System for Murine, Chimeric and Other Anti-IGF-I Receptor Antibodies 
       [0154]    The murine anti IGF-I receptor antibody was also expressed from mammalian expression plasmids similar to those used to express the humanized antibody (above). Expression plasmids are known that have murine constant regions including the light chain kappa and heavy chain gamma-1 sequences (McLean et al., 2000,  Mol. Immunol.,  37, 837-845). These plasmids were designed to accept any antibody variable region, such as for example the murine anti-IGF-I receptor antibody, by a simple restriction digest and cloning. Additional PCR of the anti-IGF-I receptor antibody was usually required to create the restriction compatible with those in the expression plasmid. 
         [0155]    An alternative approach for expressing the fully murine anti-IGF-I receptor antibody was to replace the human constant regions in the chimeric anti-IGF-I receptor antibody expression plasmid. The chimeric expression plasmid ( FIG. 16 ) was constructed using cassettes for the variable regions and for both the light and heavy chain constant regions. Just as the antibody variable sequences were cloned into this expression plasmid by restriction digests, separate restriction digests were used to clone in any constant region sequences. The kappa light chain and gamma-1 heavy chain cDNAs were cloned, for example, from murine hybridoma RNA, such as the RNA described herein for cloning of the anti-IGF-I antibody variable regions. Similarly, suitable primers were designed from sequences available in the Kabat database (see Table 10). For example, RT-PCR was used to clone the constant region sequences and to create the restriction sites needed to clone these fragments into the chimeric anti-IGF-I receptor antibody expression plasmid. This plasmid was then used to express the fully murine anti-IGF-I receptor antibody in standard mammalian expression systems such as the CHO cell line. 
         [0000]    
       
         
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Primers designed to clone the murine gamma-1 
                   
               
               
                 constant region and murine kappa constant 
               
               
                 region respectively. 
               
               
                 The primers were designed from sequences 
               
               
                 available in the Kabat database (Johnson, G  
               
               
                 nd Wu, T.T. (2001) Nucleic Acids Research, 
               
               
                 29:205-206). 
               
             
          
           
               
                 Murine Constant Region Primers 
                 SEQ 
                   
               
             
          
           
               
                 Primer 
                   
                 ID 
                   
               
               
                 name 
                 Primer Sequence 
                 NO: 
               
               
                   
               
               
                 MuIgG1 
                 TTTTGAGCTCTTATTTACCAGGAGAGTGGGAGAGGCTCT 
                 45 
                   
               
               
                 C3endX 
                 T 
               
               
                   
               
               
                 MuIgG1 
                 TTTTAAGCTTGCCAAAACGACACCCCCATCTGTCTAT 
                 46 
               
               
                 C5endH 
               
               
                   
               
               
                 MuIgKap 
                 TTTTGGATCCTAACACTCATTCCTGTTGAAGC 
                 47 
               
               
                 C3endB 
               
               
                   
               
               
                 MuIgKap 
                 TTTTGAATTCGGGCTGATGCTGCACCAACTG 
                 48 
               
               
                 C5endE 
               
               
                   
               
             
          
         
       
     
       Statement of Deposit 
       [0156]    The hybridoma that makes murine EM164 antibody was deposited with the American Type Culture Collection, PO Box 1549, Manassas, Va. 20108, on Jun. 14, 2002, under the Terms of the Budapest Treaty, and assigned deposit number PTA-4457. 
         [0157]    Certain patents and printed publications have been referred to in the present disclosure, the teachings of which are hereby each incorporated in their respective entireties by reference. 
         [0158]    While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of skill in the art that various changes and modifications can be made thereto without departing from the spirit and scope thereof.
Antibodies, humanized antibodies, resurfaced antibodies, antibody fragments, derivatized antibodies, and conjugates of these molecules with cytotoxic agents, which specifically bind to and inhibit insulin-like growth factor-I receptor, antagonize the effects of IGF-I and are substantially devoid of agonist activity toward the insulin-like growth factor-I receptor. These molecules can be conjugated to cytotoxic agents for use 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 and pancreatic cancer. These molecules can also be labeled for in vitro and in vivo diagnostic uses, such as in the diagnosis and imaging of tumors that express elevated levels of IGF-I receptor.