JP2010518140A - Novel antibody against IGF-1R - Google Patents

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R, specifically hIGF-1R. Also disclosed are antibody preparations comprising the antibodies or antigen-binding fragments of the invention. Methods for producing such antibodies or antigen-binding fragments, and their use are also within the scope of the present invention.
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Description

  The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to human insulin-like growth factor receptor (hIGF-1R). The present invention also relates to a method for treating a disease or disorder using the antibody and the antigen-binding fragment thereof, a pharmaceutical composition containing the antibody and the antigen-binding fragment thereof, and a production method.

The human insulin-like growth factor receptor (also known as IGF-1R, CD221, or EC 2.7.112) is a tyrosine kinase receptor that has 70% homology with the insulin receptor. The receptor is activated by two ligands IGF-I and IGF-II that bind with high affinity to the receptor. This receptor is a disulfide bonded αβ dimer and is denoted (αβ) ₂ . The α chain is completely extracellular, but the β chain transmembranes and has an extracellular domain and an intracellular signaling domain. Ligand-mediated receptor activation triggers intracellular events including activation of the MAPK and PI3K-protein kinase B pathways. Although it is known that IGF-1R has an important role in the growth and development of normal fetuses and postnatal infants, IGF-1R also plays an important role in cancer biology, and is known for its mitogenic, It has been implicated in several biological pathways, including conversion and protection from apoptosis (Baserga et al., (1997) Endocrine, 7 (1): 99-102, Baserga (2003) Int J Cancer, 107 ( 6): 873-7, Larsson et al., (2005) Br J Cancer, 92 (12): 2097-101, Romano (2003) Drug News Perspect, 16 (8): 525-31). Furthermore, receptor expression levels are known to be upregulated in various tumor types (reviewed by Khandwala et al. (2000) Endocr Rev., 21 (3): 215-44) and the ligand IGF-I Increased levels are associated with an increased risk of developing prostate cancer (Chan et al. (1998) Science, 279 (5350): 563-6).

  Antagonists of the IGF-1R signaling pathway are known for their anti-tumor effects in vitro and in vivo (Hofmann et al. (2005) Drug Discov Today, 10 (15): 1041-7 and Zhang et al. (2004) Expert Opin Investig Drugs, 13 (12): 1569-77). Techniques include neutralizing antibodies (Kull et al. (1983) J Biol Chem., 258 (10): 6561-6 and Li et al. (1993) Cancer Immunol Immunother., 49 (4-5): 243-52, Xiong et al., (1992) Proc Natl Acad Sci US A., 89 (12): 5356-60, Burtrum et al., (2003) Cancer Res., 63 (24): 8912-21, Cohen et al., (2005) Clin Cancer. Res., 11 (5): 2063-73, Maloney et al., (2003) Cancer Res., 63 (16): 5073-83, Jackson-Booth et al., (2003) Horm Metab Res., 35 (11-12) : 850-6), antisense (Resnicoff et al., (1994) Cancer Res., 54 (18): 4848-50, Lee et al., (1996) Cancer Res., 56 (13): 3038-41, Muller (1998) Int J Cancer., 77 (4): 567-71, Trojan et al. (1993) Science, 259 (5091): 94-7, Shapiro et al. (1994) J Clin Invest., 94 (3 ): 1235-42), dominant negative mutants (Prager et al. (1994) Proc Natl Acad Sci US A., 91 (6): 2181-5), and small molecule tyrosine kinase inhibitors (Hopfner et al., (2006) Biochem Pharmacol. 2006, 71 (10): 1435-48), and IGF binding protein (IGFB) P-Nickerson et al. (1997) Biochem Biophys Res Commun., 237 (3): 690-3). Known monoclonal antibodies include WO99 / 60023, WO03 / 100008, WO02 / 053596, WO04 / 071529, European Patent 0629240, WO03 / 059951, WO03 / 106621, WO04 / 083248, WO04 / 087756, US2006452167A, and the like. However, there is a need for antibodies with improved effector function, such as antibodies with improved ADCC and / or CDC function.

  The structure of an antibody is well known in the art, and in particular, the heavy chain constant region is known to have a glycosylated sugar chain, which is an N-glycoside-linked sugar chain such as N-glycosides. It can be acetlyglucosamine and the sugar chain may or may not be fucosylated.

  Methods for measuring fucosylation levels are well known in the art, for example, for antibody populations, acid hydrolysis is used to remove glycosylated sugar chain monosaccharides from antibodies. These can be labeled with a dye such as 2-aminobenzoic acid (2-AA). A calibration curve can then be generated to perform reverse phase high performance liquid chromatography with fluorescence detection and to quantify the sample. The ratio of fucose to mannose per antibody population can then be calculated.

  In one embodiment, according to the invention, an antibody or antigen binding thereof that specifically binds to IGF-1R, comprising CDR H3 of SEQ ID NO: 1, or a variant thereof comprising one or two amino acid substitutions in CDR H3 Fragments are provided.

  In one embodiment, according to the present invention, an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R, particularly hIGF-1R and neutralizes the activity of hIGF-1R, comprising CDR H3 of SEQ ID NO: 1 Or an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising a variant thereof wherein one or two amino acid residues in CDR H3 differ from the amino acid residue at its corresponding position in SEQ ID NO: 1 The

  Also provided are methods of making the antibodies described herein, comprising the step of expressing the antibody or antigen-binding fragment thereof in a cell line.

  In another embodiment, a kit of parts is provided that includes instructions for use with the compositions described herein.

  Also provided is a method of treating a human patient suffering from cancer comprising administering a therapeutically effective amount of the antibody preparation described herein.

It is a figure which shows the coupling | bonding of the purified mouse monoclonal antibody and human IGF-1R which were measured by ELISA. It is a figure which shows the coupling | bonding of the purified 6E11 chimeric antibody and 6E11 humanized antibody, and human IGF-1R which were measured by ELISA. In FIG. 2A, the H0L0 binding curve shifted to the right because the antibody concentration was very low and could not be accurately quantified. On the other hand, in FIG. 2D, the overall trend was similar, but the signal decreased compared to the other assays. It is a figure which shows the coupling | bonding of the purified 6E11 chimeric antibody and 6E11 humanized antibody, and human IGF-1R which were measured by ELISA. It is a figure which shows the coupling | bonding of the purified 6E11 chimeric antibody and 6E11 humanized antibody, and human IGF-1R which were measured by ELISA. It is a figure which shows the coupling | bonding of the purified 6E11 chimeric antibody and 6E11 humanized antibody, and human IGF-1R which were measured by ELISA. It is a figure which shows the coupling | bonding of the purified 6E11 chimeric antibody and 6E11 humanized antibody, and human IGF-1R which were measured by ELISA. FIG. 3 shows that IGF-1R receptor is downregulated after incubation of 3T3 / LISN c4 cells with purified 6E11 mouse monoclonal antibody against human IGF-1R for 24 hours. FIG. 5 shows that IGF-1R receptor is downregulated after incubation of NCI-H838 cells with humanized H0L0 antibody against human IGF-1R for up to 24 hours. FIG. 7 shows that IGF-1R receptor and insulin receptor are down-regulated after incubation of NCI-H838 cells with H0L0, H0L0 IgG1m (AA), or non-targeted human IgG for 24 hours. FIG. 6 is a histogram showing the fluorescence intensity of IGF-1R levels of granulocyte and lymphocyte populations after incubation with H0L0 at 4 ° C. and 37 ° C. FIG. FIG. 6 is a histogram of fluorescence intensity showing IGF-1R levels of granulocyte and lymphocyte populations after incubation with H0Lo at 4 ° C. and 37 ° C. compared to isotype controls. FIG. 5 shows receptor phosphorylation inhibition mediated by purified mouse monoclonal antibodies 6E11, 5G4, and 15D9. FIG. 3 shows an example of receptor phosphorylation inhibition mediated by H1L0 compared to 6E11c. FIG. 6 shows examples of receptor phosphorylation inhibition mediated by H0L0 and H0L0 IgG1m (AA) and H1L0 and H10L0 IgG1m (AA). FIG. 11A shows examples of the activity of various purified mouse monoclonal antibodies in a competitive ELISA. FIG. 11B: shows an example of H1L0 activity in a competitive ELISA compared to 6E11c. A competitive ELISA to demonstrate the ability of purified 6E11 mouse monoclonal antibody or 6E11 chimeric antibody or 6E11 humanized antibody to inhibit the binding of IGF-1R receptor to a second neutralizing antibody. A competitive ELISA to demonstrate the ability of purified 6E11 mouse monoclonal antibody or 6E11 chimeric antibody or 6E11 humanized antibody to inhibit the binding of IGF-1R receptor to a second neutralizing antibody. A competitive ELISA to demonstrate the ability of purified 6E11 mouse monoclonal antibody or 6E11 chimeric antibody or 6E11 humanized antibody to inhibit the binding of IGF-1R receptor to a second neutralizing antibody. FIG. 13A: Binding of purified mouse monoclonal antibody and recombinant cynomolgus monkey IGF-1R quantified by ELISA. FIG. 13B: Binding of purified humanized monoclonal antibody and recombinant cynomolgus monkey IGF-1R compared to 6E11 chimera (6E11c). FIG. 2 shows an insulin receptor binding ELISA using purified mouse monoclonal antibody. FIG. 5 shows an insulin receptor binding ELISA using purified humanized antibody. FACS assay demonstrating that this antibody recognizes the Colo205 tumor cell line. FACS assay demonstrating that this antibody recognizes the NCI-H838 lung cancer tumor cell line. FACS assay demonstrating that this antibody recognizes MCF7 carcinoma and A549 lung carcinoma cell lines. FIG. 6 shows immunohistochemistry of frozen tissue samples of tumor and normal prostate samples using purified mouse monoclonal antibodies. FIG. 6 shows immunohistochemistry of frozen tissue samples of tumor breast samples using purified mouse monoclonal antibodies. FIG. 10 shows immunohistochemistry of frozen tissue samples of tumor breast samples using purified H1L0 humanized and 6E11 chimeric monoclonal antibodies. FIG. 5 shows immunohistochemistry of frozen tumor tissue samples of tumor breast samples using biotin-labeled H0L0 antibody. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified mouse monoclonal antibody. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified H1L0 humanized or 6E11 chimeric monoclonal antibodies. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified humanized or purified mouse 6E11 monoclonal antibody. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified humanized or purified mouse 6E11 monoclonal antibody. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified humanized or purified mouse 6E11 monoclonal antibody. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified humanized or purified mouse 6E11 monoclonal antibody. FIG. 5 shows inhibition of IGF-I mediated proliferation of 3T3 / LISN c4 cells inhibited by purified humanized or purified mouse 6E11 monoclonal antibody. FIG. 5 shows inhibition of IGF-I mediated cell cycle by purified mouse monoclonal antibody quantified by propidium iodide staining and flow cytometry. FIG. 6 shows reversal of IGF-1-mediated protection from camptothecin-induced apoptosis in NCI-H838 cells by mouse 6E11 monoclonal antibody. FIG. 5 shows reversal of IGF-1-mediated protection from camptothecin-induced apoptosis in A549 cells by selected antibodies. It is a figure which shows that the agonist activity of a purified mouse monoclonal antibody does not exist in presence of a bridge | crosslinking antibody. FIG. 5 shows the activity of H0L0 antibody on basal levels of phospho-AKT in differentiated preadipocytes compared to negative control antibody. FIG. 5 shows the activity of H0L0 antibody on basal levels of phosphor-AKT in A549 cells. FIG. 3 shows the activity of humanized antibodies on basal IGF-1R receptor phosphorylation levels in 3T3 / LISN c4 cells. FIG. 3 shows the activity of cross-linked humanized antibodies on basal IGF-1R receptor phosphorylation levels in 3T3 / LISN c4 cells. FIG. 3 shows the activity of humanized antibodies on the growth of NCI-H929 cells in the absence of ligand stimulation. FIG. 4 shows inhibition of 3T3 / LISN c4 tumor growth after treatment with 6E11 monoclonal antibody in nude mice. FIG. 4 shows inhibition of 3T3 / LISN c4 tumor growth after treatment with 6E11 monoclonal antibody in nude mice. FIG. 4 shows inhibition of 3T3 / LISN c4 tumor growth after treatment with 6E11 and humanized monoclonal antibodies in nude mice. FIG. 5 shows inhibition of Colo205 tumor growth after treatment with 6E11 monoclonal antibody in nude mice. FIG. 5 shows receptor binding kinetics using NCI-H838 cells incubated with H0L0. IGF-1R / IR heterodimer binding assay using Colo-205 cells and recombinant NIH-3T3 cells for 6E11, 6E11c, and H0L0 mIgG (AA) antibodies. FIG. 6 shows the growth of NCI-H929 cells in the presence of 6E11 and H0L0. It is a figure which shows the measurement of stability of H0L0 in serum. FIG. 5 shows in vivo downregulation of IGR-1R in LISN / 3T3 c4 cells after H0L0 or 6E11 treatment.

Detailed Description of the Invention

BRIEF DESCRIPTION OF THE TABLES Table 1: SEQ ID NOs of hybridoma variable heavy and light chains.
Table 2: IC50 values of selected antibodies in phosphorylation assays.
Table 2a: IC50 values of selected antibodies in phosphorylation assay.
Table 3: Kinetic data for mouse monoclonal antibodies.
Table 4: Kinetic data for humanized monoclonal antibodies.
Table 5: Kinetic data for humanized monoclonal antibodies of wild type Fc and invalidated Fc.
Table 6: Kinetic data for humanized monoclonal antibodies.
Table 7: Kinetic data for humanized monoclonal antibodies.
Table 8: Kinetic data comparing anti-IGF-1R to human and cynomolgus monkey IGF-1R.
Table 9: Inhibition values on the IGF-1 surface of 200 RU.
Table 10: 4000RU IGF-1 surface inhibition values.
Table 11: Neutralization of receptor-ligand binding.
Table 12: Summary of immunohistochemical analysis of tumor tissue microarray.
Table 13: Activity of various antibodies in AKT phosphorylation assay.
Table 14: Activity of various antibodies in the AKT phosphorylation assay.

  The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R, eg, specifically binds to hIGF-1R.

  In one embodiment of the invention, an antibody or antigen-binding fragment thereof that specifically binds to hIGF-1R and neutralizes the activity of hIGF-1R, comprising CDR H3 of SEQ ID NO: 1, or CDR H3 An antibody or antigen-binding fragment thereof that specifically binds to IGF-1R, comprising a heavy chain variable domain comprising a variant thereof wherein one or two amino acid residues are different from the corresponding amino acid residues of SEQ ID NO: 1 Is provided.

  In one embodiment of the invention, these differences in amino acid residues are conservative substitutions.

  Another embodiment of the invention comprises CDR H3 that specifically binds to IGF-1R and is a variant of the sequence shown in SEQ ID NO: 1, wherein one or two of the variants CDRH3 Residue is different from the residue at the corresponding position of position 7 and / or position 9 of SEQ ID NO: 1 (in which case the first residue is W at position 1 and the last residue V is at position 14) An antibody or antigen-binding fragment thereof is provided.

  Another embodiment of the invention specifically comprises CDR H3, which binds specifically to IGF-1R and is a variant of the sequence shown in SEQ ID NO: 1, wherein one or two residues in the CDR H3 of the variant A group by substitution of R at position 7 with S, or by substitution of K at position 9 with R, or with substitution of R at position 7 with S and K in position 9 with R; An antibody or antigen-binding fragment thereof that differs from the residue at its corresponding position is provided.

  In another embodiment of the present invention, the following sequences are further included: CDR H2 set forth in SEQ ID NO: 2, CDR H1 set forth in SEQ ID NO: 3, CDR L1 set forth in SEQ ID NO: 4, CDR L2 set forth in SEQ ID NO: 5 And an antigen-binding fragment thereof comprising one or more of CDR L3 set forth in SEQ ID NO: 6.

  In one embodiment of the invention there is provided an antibody or antigen-binding fragment thereof wherein CDR H1, H2, and H3, and CDR L1 and L3 are derived from 6E11 and CDR L2 is derived from 9C7.

  In one embodiment of the invention, one or more of the CDRs of the antibody or antigen-binding fragment thereof may comprise a variant of CDR shown in the sequence above. Each variant CDR will contain one or two amino acid residues that differ from the corresponding amino acid residue in the sequence. Such substitution of amino acid residues can be, for example, conservative substitutions such as substituting one hydrophobic amino acid with another hydrophobic amino acid, eg, substituting leucine for valine or isoleucine.

  In another embodiment of the invention, comprising CDR H3 and further comprising the following sequences: CDR H2 (SEQ ID NO: 2), CDR H1 (SEQ ID NO: 3), CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7) And an antibody or antigen-binding fragment thereof comprising one or more of CDR L3 (SEQ ID NO: 6).

  In yet another embodiment of the invention, comprising CDR H3 and further comprising the following sequences: CDR H2 (SEQ ID NO: 2), CDR H1 (SEQ ID NO: 3), CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7 ), And an antigen-binding fragment thereof comprising one or more of CDR L3 (SEQ ID NO: 6), wherein one or more of the CDRs has one or two amino acid substitutions in each variant CDR It may be replaced by a mutant containing.

  In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises CDR H3 of SEQ ID NO: 1 and CDR H1 of SEQ ID NO: 3. In another embodiment, the antibody or antigen-binding fragment thereof comprises CDR H3 of SEQ ID NO: 1 and CDR L2 of SEQ ID NO: 7. In yet another embodiment, an antibody or antigen-binding fragment thereof of the invention comprises CDR H3 of SEQ ID NO: 1, CDR H1 of SEQ ID NO: 3, and CDR L2 of SEQ ID NO: 7.

In another embodiment of the invention, there is provided an antibody or antigen-binding fragment thereof according to the invention described herein, further comprising the following CDRs or antigen-binding fragments thereof:
CDR H1 (SEQ ID NO: 3)
CDR H2 (SEQ ID NO: 2)
CDR H3 (SEQ ID NO: 1)
CDR L1 (SEQ ID NO: 4)
CDR L2 (SEQ ID NO: 7)
CDR L3 (SEQ ID NO: 6).

  In another embodiment of the invention, there is provided an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R and comprises a CDR that is a variant of the above sequence.

  In another embodiment of the invention, it specifically binds to IGF-1R and is a heavy chain variable domain of SEQ ID NO: 8 and a light chain variable domain of SEQ ID NO: 9, or a heavy chain variable domain of SEQ ID NO: 10 and SEQ ID NO: 11 light chain variable domains, or heavy chain variable domain of SEQ ID NO: 12 and light chain variable domain of SEQ ID NO: 13, or heavy chain variable domain of SEQ ID NO: 14 and light chain variable domain of SEQ ID NO: 16, or SEQ ID NO: 15 An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain and a light chain variable domain of SEQ ID NO: 16 is provided.

  In another embodiment of the invention there is provided an isolated heavy chain variable domain of an antibody comprising SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15, eg, SEQ ID NO: 12.

  In another embodiment of the present invention, an antibody or antigen-binding fragment thereof comprising a CDR according to the present invention as described herein, or a heavy or light chain variable domain according to the present invention as described herein. Rats, mice, primates (eg, cynomolgus monkeys, Old World monkeys, or apes) or human antibodies or antigen-binding fragments thereof are provided.

  In another embodiment of the invention, the antibody or antigen-binding fragment thereof described herein further binds to a primate IGF-1R, such as Cynomolgus macaque IGF-1R.

  In another embodiment of the invention, in the context of a human framework, for example as a humanized or chimeric antibody the following CDRs: CDR H3 set forth in SEQ ID NO: 1, CDR H2 set forth in SEQ ID NO: 2, set forth in SEQ ID NO: 3 There is provided an antibody or antigen-binding fragment thereof comprising one or more of the CDR H1, the CDR L1 set forth in SEQ ID NO: 4, the CDR L2 set forth in SEQ ID NO: 5, and the CDR L3 set forth in SEQ ID NO: 6.

  In one embodiment of the invention, the humanized heavy chain variable domain comprises the CDRs shown in SEQ ID NOs: 1-3 within the acceptor antibody framework, wherein the acceptor antibody framework contains the human acceptor sequence of SEQ ID NO: 59. In contrast, it has more than 80% identity in the framework region, or more than 85%, or more than 90%, or more than 95%, or more than 98%, or more than 99% identity in the framework region.

  In one embodiment of the invention, the humanized light chain variable domain comprises the CDRs shown in SEQ ID NOs: 4-6 within the acceptor antibody framework, the acceptor antibody framework comprising the human acceptor sequence of SEQ ID NO: 60. In contrast, it has more than 80% identity in the framework region, or more than 85%, or more than 90%, or more than 95%, or more than 98%, or more than 99% identity in the framework region.

  In SEQ ID NO: 59 and SEQ ID NO: 60, the position of the CDR sequence is indicated by Xaa.

  In one embodiment of the invention, an antibody or antigen-binding fragment thereof comprising a CDR according to the invention as described herein, or a heavy or light chain variable domain according to the invention as described herein, The antibody or antigen-binding fragment thereof is provided wherein the half-life of the antibody is at least 5 days, or at least 7 days or at least 9 days in a mouse animal model.

  In another embodiment of the present invention, an antibody or antigen-binding fragment thereof comprising a CDR according to the present invention as described herein, or a heavy or light chain variable domain according to the present invention as described herein. Further provided is an antibody or antigen-binding fragment thereof, wherein the antibody further comprises a constant region that can be of any isotype or subclass. In one embodiment, the heavy chain constant region is of an IgG isotype, eg, IgG1, IgG2, IgG3, IgG4 or variants thereof. In one embodiment, the antibody is IgG1.

  In one embodiment of the invention, an antibody according to the invention as described herein comprising a constant region so as to reduce ADCC and / or complement activation or effector functionality of the antibody Is provided. In such embodiments, the heavy chain constant region can comprise a naturally disabled constant region of the IgG2 or IgG4 isotype, or a variant IgG1 constant region. A suitable modification is described in EP 0307434. An example is the substitution of alanine residues at positions 235 and 237 (EU index number).

  In another embodiment of the invention, there is provided an antibody according to the invention as described herein that can have at least some effector function, for example, can have some ADCC and / or CDC function. The In one embodiment of the invention, an antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region, which specifically binds to IGF-1R, eg, human IGF-1R, and comprises CDR H3 of SEQ ID NO: 1. Or an antigen-binding fragment thereof comprising a variant thereof comprising one or two amino acid substitutions in CDR H3, eg, an antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region, comprising CDRs Selected from H1 (SEQ ID NO: 3), CDR H2 (SEQ ID NO: 2), CDR H3 (SEQ ID NO: 1), CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7), and CDR L3 (SEQ ID NO: 6) Of an IgG1 wild type, IgG2 wild type, IgG3 wild type, IgG4 wild type, or an enhanced form thereof Antibody or antigen-binding fragment thereof comprising a constant region are provided.

  In one embodiment of the invention, the antibody described herein comprising a constant region or an antigen-binding fragment thereof linked to a constant region is selected from IGF-1R, EGFR, HER-2, or HER-3. It binds specifically to growth factor receptors. For example, an antibody that specifically binds to HER-2 or HER-3, or an antibody that specifically binds to, for example, IGF-1R or EGRF, eg, human IGF-1R.

  In one embodiment, the antibodies of the invention are linked to one or more domain antibodies having specificity for VEGF or EGFR.

  In one embodiment of the invention, an antibody according to the invention described herein comprising one or more mutations in its heavy chain constant region such that the antibody or antigen-binding fragment has an enhanced effector function Or an antigen-binding fragment thereof is provided. For example, it has enhanced ADCC or enhanced CDC, or it has enhanced ADCC and CDC effector functions. Suitable modifications are described in Shields et al., J. Biol. Chem (2001) 276: 6591-6604, Lazar et al., PNAS (2006) 103: 4005-4010, and US Pat. Has been.

  In one embodiment of the invention, an antibody comprising a heavy chain constant region or an antigen-binding fragment thereof linked to a heavy chain constant region, wherein the antibody specifically binds to IGF-1R, eg, human IGF-1R, or a An antigen binding fragment is provided. The antibody or antigen-binding fragment thereof comprises CDR H3 of SEQ ID NO: 1, or a variant thereof in which one or two amino acid residues in CDR H3 differ from the amino acid residue at its corresponding position in SEQ ID NO: 1. Well, and compared to the wild type, the antibody or antigen-binding fragment thereof includes a heavy chain constant region that has been mutated to have enhanced effector function. For example, an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R and comprises CDR H3 of SEQ ID NO: 1, such as CDR H1 (SEQ ID NO: 3), CDR H2 (SEQ ID NO: 2), CDR H3 (SEQ ID NO: 1) a CDR selected from CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7), and CDR L3 (SEQ ID NO: 6), and compared to the wild type, the antibody or antigen-binding fragment thereof An antibody or antigen-binding fragment thereof comprising a heavy chain constant region mutated to have enhanced effector function.

  In one embodiment of the invention, such mutations are in one or more of the positions selected from 239, 332, and 330 (IgG1), or in equivalent positions of other IgG isotypes. Examples of suitable mutations are S239D and I332E and A330L. In one embodiment, the antibody or antigen-binding fragment is mutated at positions 239 and 332, such as S239D and I332E, such as 239 and 332 and 330, such as 3 or more positions selected from S239D and I332E and A330L. It has been mutated.

  In another embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, comprising SEQ ID NO: 64 and SEQ ID NO: 66 An antibody or antigen-binding fragment thereof comprising a constant region selected from the constant regions set forth in, for example, the heavy chain constant region shown in SEQ ID NO: 64 or SEQ ID NO: 66 together with the variable domains of SEQ ID NO: 14 and SEQ ID NO: 15 An antibody or antigen-binding fragment, eg, an antibody comprising a heavy chain constant region or an antigen-binding fragment thereof linked to a heavy chain constant region, comprising an antibody or antigen thereof comprising SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 64 A binding fragment is provided. In another embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, comprising SEQ ID NO: 64 and SEQ ID NO: 66 An antibody or antigen-binding fragment thereof comprising a heavy chain constant region selected from the heavy chain constant regions shown in FIG. 1, for example, the heavy chain constant shown in SEQ ID NO: 64 or SEQ ID NO: 66 together with the variable domains of SEQ ID NO: 14 and SEQ ID NO: 16 An antibody or antigen-binding fragment thereof comprising a normal region, for example, an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 64 is provided.

  In one embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, wherein the antibody or antigen-binding fragment is enhanced An antibody or antigen-binding fragment thereof comprising a heavy chain constant region with a modified glycosylation profile so as to have a modified effector function is provided. For example, the antibody or antigen-binding fragment has enhanced ADCC or enhanced CDC, or has enhanced ADCC and CDC effector function. Examples of suitable methods for producing antibodies with altered glycosylation profiles are described in WO2003011878, WO2006014679 and EP1229125.

  In one embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention as described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, comprising IGF-1R, eg human IGF- Antibodies or antigen-binding fragments thereof that specifically bind 1R are provided. The antibody or antigen-binding fragment thereof may comprise CDR H3 of SEQ ID NO: 1, or a variant thereof in which one or two amino acid residues in CDR H3 differ from the amino acid residue at its corresponding position in SEQ ID NO: 1. And a heavy chain constant region in which the glycosylation profile is modified such that the antibody or antigen-binding fragment has enhanced effector function compared to the wild type.

  For example, an antibody or antigen-binding fragment thereof that specifically binds to IGF-1R, eg, human IGF-1R, and comprises CDR H3 of SEQ ID NO: 1, such as CDR H1 (SEQ ID NO: 3), CDR H2 (SEQ ID NO: 2) An antibody comprising a CDR selected from CDR H3 (SEQ ID NO: 1), CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7), and CDR L3 (SEQ ID NO: 6) and compared to the wild type Alternatively, an antibody or antigen-binding fragment thereof comprising a heavy chain constant region whose glycosylation profile has been modified so that the antigen-binding fragment has an enhanced effector function.

  In one embodiment, the invention provides that the ratio of fucose to mannose in the antibody preparation is 0.8: 3 or less, such as 0.7: 3 or less, or 0.6: 3 or less, or 0.5: Antibody preparations are provided that are 3 or less, or 0.4: 3 or less, or 0.3: 3 or less, or 0.2: 3 or less, or 0.1: 3 or less. In one embodiment, the antibody preparation contains no or negligible bound fucose.

  In another embodiment of the invention, an antibody preparation comprising an antibody or antigen-binding fragment thereof comprising the variable domains of SEQ ID NO: 14 and SEQ ID NO: 15 or SEQ ID NO: 14 and SEQ ID NO: 16, wherein the antibody preparation comprises The ratio of fucose to mannose is 0.8: 3 or less, such as 0.7: 3 or less, or 0.6: 3 or less, or 0.5: 3 or less, or 0.4: 3 or less, or 0.3: Antibody preparations are provided that are 3 or less, or 0.2: 3 or less, or 0.1: 3 or less. In one embodiment, the antibody preparation contains no or negligible bound fucose.

  In one embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, wherein the antibody or antigen-binding fragment is enhanced. An antibody or antigen-binding fragment thereof comprising a heavy chain constant region mutated to have an effector function and an altered glycosylation profile, eg, one of the following functions: enhanced ADCC or enhanced CDC Antibodies or antigen-binding fragments thereof are provided that have a plurality, eg, have enhanced ADCC function.

  In one embodiment of the invention, an antibody preparation comprising an antibody comprising an immunoglobulin heavy chain constant region described herein or an antigen-binding fragment thereof linked to an immunoglobulin heavy chain constant region, comprising the immunoglobulin The heavy chain constant region provides effector function to the antibody or antigen-binding fragment, and the antibody or antigen-binding fragment specifically binds to a growth factor receptor, and the immunoglobulin heavy chain constant region has at least two And the glycosylation profile is altered so that the ratio of fucose to mannose is 0.8: 3 or less, so that the antibody or antigen-binding fragment is free of the mutation, An equivalent antibody or antigen-binding fragment having an immunoglobulin heavy chain constant region with an unmodified glycosylation profile With effector function enhanced as compared with the segment, antibody preparations are provided. The altered glycosylation profile of the antibody preparation is not the result of the immunoglobulin heavy chain mutation.

  For example, such an antibody or antigen-binding fragment specifically binds to IGF-1R, eg, human IGF-1R, and comprises CDR H3 of SEQ ID NO: 1, for example, the antibody or antigen-binding fragment comprises CDR H1 ( CDR selected from SEQ ID NO: 3), CDR H2 (SEQ ID NO: 2), CDR H3 (SEQ ID NO: 1), CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7), and CDR L3 (SEQ ID NO: 6) And the mutated heavy chain constant region so that the antibody or antigen-binding fragment thereof has enhanced effector function and has a modified glycosylation profile. For example, such an antibody or antigen-binding fragment can comprise the variable domains of SEQ ID NO: 14 and SEQ ID NO: 15 or SEQ ID NO: 14 and SEQ ID NO: 16.

  In such embodiments, the mutation is at one or more positions selected from 239, 332 and 330 (IgG1), or equivalent positions of other IgG isotypes. Examples of suitable mutations are S239D and I332E and A330L. In one embodiment, an antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region has a mutation at 239 and 332, for example, S239D and I332E, or even 239, 332, and 330, for example, Mutations may be included at three or more positions selected from S239D and I332E and A330L.

  In one embodiment, the ratio of fucose to mannose in the antibody preparation is 0.8: 3 or less, such as 0.7: 3 or less, or 0.6: 3 or less, or 0.5: 3 or less, Or 0.4: 3 or less, or 0.3: 3 or less, or 0.2: 3 or less, or 0.1: 3 or less. In one embodiment, the antibody preparation contains no or negligible bound fucose.

  In one embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, comprising a chimeric heavy chain constant region, For example, the antibody or antigen-binding fragment comprises at least one CH2 domain from IgG3 such that it has an enhanced effector function, such as having one or more of the following functions: enhanced ADCC or enhanced CDC For example, an antibody or antigen-binding fragment having enhanced CDCC is provided. For example, an antibody or antigen-binding fragment can comprise one CH2 domain from IgG3 or both CH2 domains from IgG3.

In another embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, wherein the variant and chimeric heavy chain constants A region, for example, comprising at least one CH2 domain from IgG3 and one CH2 domain from IgG1, wherein the CH2 domain of IgG1 is 1 or at a position selected from 239, 332, and 330 The mutation is selected from S239D, I332E, and A330L such that the antibody has an enhanced effector function, eg, the following functions: enhanced ADCC or enhanced CDC An antibody having one or more, for example having enhanced ADCC and enhanced CDCC, or Antigen-binding fragment is provided. In one embodiment, the IgG1 CH ₂ domain has mutations S239D and I332E.

  In one embodiment of the invention, an antibody comprising a heavy chain constant region according to the invention described herein or an antigen-binding fragment thereof linked to a heavy chain constant region, wherein the heavy chain constant region is from IgG3. Includes a chimeric heavy chain constant region and modified glycosylation profile to include at least one CH2 domain and one CH2 domain from IgG1, and a fucose to mannose ratio of 0.8: 3 or less An immunoglobulin heavy chain antibody that does not have the mutation and modified glycosylation profile such that the antibody or antigen-binding fragment has an enhanced effector function. The antibody or antigen-binding fragment is enhanced compared to an equivalent antibody or antigen-binding fragment having a normal region. There is provided an antibody or antigen-binding fragment having an effector function, eg, having one or more of the following functions: enhanced ADCC or enhanced CDC, eg, enhanced ADCC and enhanced CDCC The

  In another embodiment, the antibody or antigen-binding fragment has at least one IgG3 CH2 domain and at least one heavy chain constant domain from IgG1, and according to the limitations described herein, both IgG CH2 domains are It has been mutated.

  In one embodiment of the invention, an antibody preparation comprising an antibody comprising a heavy chain constant region or an antigen-binding fragment thereof linked to a heavy chain constant region, comprising a variant and a chimeric heavy chain constant region comprising: The glycosylation profile of the antibody preparation has been modified so that the antigen-binding fragment has an enhanced effector function, eg, one or more of the following functions: enhanced ADCC or enhanced CDC. An antibody preparation is provided. In one embodiment, the mutation is selected from positions 239 and 332 and 330, by way of example, the mutation is selected from S239D and I332E and A330L. In another embodiment, the heavy chain constant region comprises at least one CH2 domain from IgG3 and one CH2 domain from IgG1. In one embodiment, the heavy chain constant region glycosylation profile is modified such that the ratio of fucose to mannose is 0.8: 3 or less, so that the mutation is absent and the glycosylation profile is Compared to an equivalent non-chimeric antibody or antigen-binding fragment thereof having an immunoglobulin heavy chain constant region that has not been modified, said antibody or antigen-binding fragment has an enhanced effector function.

  In one embodiment of the invention, there is provided a recombinantly transformed, transfected or transduced host cell comprising at least one expression cassette, for example an expression cassette according to the invention described herein. A polynucleotide encoding the heavy chain of an antibody or antigen-binding fragment thereof, and further comprising a polynucleotide encoding the light chain of an antibody or antigen-binding fragment thereof according to the invention described herein, or two There is an expression cassette, the first expression cassette encodes the light chain and the second expression cassette encodes the heavy chain. For example, in one embodiment, the first expression cassette comprises a polynucleotide encoding the heavy chain of an antibody comprising a constant region according to the invention described herein or an antigen-binding fragment thereof linked to the constant region. A second cassette comprising a polynucleotide encoding a light chain of an antibody comprising a constant region according to the invention described herein or an antigen-binding fragment thereof linked to the constant region, for example, comprising: The expression cassette comprises a polynucleotide encoding a heavy chain selected from SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 67, or SEQ ID NO: 70, and the second expression cassette is selected from SEQ ID NO: 42 or SEQ ID NO: 69. A polynucleotide encoding the light chain.

  Another embodiment of the invention comprises one or more expression cassettes encoding the heavy and / or light chains of an antibody comprising the constant regions described herein or antigen-binding fragments thereof linked to the constant regions. A stably transformed host cell comprising the vector is provided. For example, such a host cell may include a first vector encoding a light chain and a second vector encoding a heavy chain, eg, the first vector is SEQ ID NO: 37, SEQ ID NO: 38 or the sequence The second vector encodes a light chain, eg, the light chain of SEQ ID NO: 39, encoding a heavy chain selected from number 68.

  In another embodiment of the invention, there is provided a host cell according to the invention as described herein, wherein the cell is a eukaryote, eg, the cell is a mammal. Examples of such cell lines include CHO or NS0.

  In another embodiment of the present invention, a method for producing an antibody comprising a constant region according to the present invention as described herein or an antigen-binding fragment thereof linked to a constant region, said medium comprising, for example, serum free A method is provided that includes culturing host cells in a culture medium.

  A method of making an antibody as described herein comprising the step of expressing an antibody or antigen-binding fragment thereof in a cell line, wherein the N-glycoside binds to fucose and a molecule having an immunological function thereof Methods are also provided that are adapted to modulate the presence or absence of linkage to a linking sugar chain.

  In another embodiment of the invention, there is provided a method according to the invention as described herein, wherein the antibody is further purified to at least 95% or more (eg 98% or more) with respect to antibody-containing serum-free culture medium. The

  In another embodiment of the invention, there is provided a pharmaceutical composition comprising an antibody comprising a constant region according to the invention described herein or an antigen-binding fragment thereof linked to a constant region, and a pharmaceutically acceptable carrier. Is done.

  In another embodiment of the present invention, a kit of parts is provided that includes instructions for use with the compositions of the present invention described herein.

  In another embodiment of the invention, a method of treating a human patient suffering from rheumatoid arthritis, comprising an antibody comprising a constant region according to the invention as described herein or an antigen binding thereof linked to a constant region A method is provided comprising administering a therapeutically effective amount of the fragment. An antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region can be combined with a pharmaceutically acceptable carrier.

  In another embodiment of the invention, a method of treating a human patient suffering from cancer, comprising an antibody comprising a constant region according to the invention as described herein or an antigen-binding fragment thereof linked to a constant region A method comprising the step of administering a therapeutically effective amount of An antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region can be combined with a pharmaceutically acceptable carrier.

  In another embodiment of the invention, a method of treating a human patient suffering from diabetic retinopathy, comprising an antibody comprising a constant region according to the invention described herein or a constant region linked thereto. A method is provided comprising administering a therapeutically effective amount of an antigen binding fragment. An antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region can be combined with a pharmaceutically acceptable carrier.

  In another embodiment of the invention, a method of treating a human patient suffering from macular degeneration, comprising an antibody comprising a constant region according to the invention as described herein or an antigen thereof linked to a constant region A method is provided comprising administering a therapeutically effective amount of the binding fragment. An antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region can be combined with a pharmaceutically acceptable carrier.

  In another embodiment of the invention, a method of treating a human patient suffering from cancer, comprising an antibody comprising a constant region according to the invention as described herein or an antigen-binding fragment thereof linked to a constant region And administering a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

  In another embodiment of the invention, a neovascular disease, eg, a disease or disorder selected from the group consisting of proliferative diabetic retinopathy, neovascular glaucoma and age-related macular degeneration (AMD), and the group consisting of: There is provided the use of an antibody comprising a constant region according to the invention described herein or an antigen-binding fragment thereof linked to a constant region in the manufacture of a medicament for treating a disease or disorder selected from: Rheumatoid arthritis, psoriasis, or cancer such as acute lymphoblastic leukemia, adrenocortical cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, colorectal cancer, basal cell carcinoma , Extrahepatic bile duct cancer, bladder cancer, osteosarcoma / malignant fibrous histiocytoma bone cancer, brain tumor (eg brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, upper garment Cystoma, medulloblastoma, supratent undifferentiated neuroectodermal tumor, visual tract and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, cancer of unknown primary, primary Central nervous system, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer Cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma eye cancer, retinoblastoma Cell carcinoma ocular cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor (eg extracranial, extragonadal, and ovary), gestational trophoblastic tumor, glioma (Eg adults, children Brainstem, childhood cerebral astrocytoma, childhood visual tract and hypothalamus), hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamus and visual tract glioma, Intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney (renal cell) cancer, laryngeal cancer, leukemia (eg, acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, And hair cells), lip and oral cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma (eg AIDS related, Burkitt, skin T cells, Hodgkin, non-Hodgkin, and primary central nervous system), Waldenstrom's macroglobulinemia, bone malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous with unknown primary Cervical cancer, multiple endocrine neoplasia symptoms Multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, chronic myeloproliferative disorder, Nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian hypoplastic potential Tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineoblastoma, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma , Primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary Syndrome, non-melanoma skin cancer, Merkel cell skin carcinoma, small intestine cancer, Tissue sarcoma, squamous cell carcinoma, cutaneous T-cell lymphoma, testicular cancer, thymoma, thymic cancer, thyroid cancer, gestational trophoblastic tumor, carcinoma of unknown primary site, cancer of unknown primary site, urethral cancer, endometrial uterine cancer, Uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

  In another embodiment of the invention, the patient has proliferative diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, rheumatoid arthritis, psoriasis, colorectal cancer, breast cancer, prostate cancer, lung cancer, or myeloma There is provided a method according to the invention as described herein suffering from one or more of the following:

The term “antibody” is used in a broad sense in the present specification, and specifically, a monoclonal antibody (such as a full-length monoclonal antibody), a polyclonal antibody, a multispecificity, as long as it exhibits a desired biological activity. Includes antibodies (eg, bispecific antibodies), and antibody fragments. These will be described in more detail below.

  The term “monoclonal antibody” as used herein refers to a substantially homogeneous population of antibodies, ie, individual mutations that are included in the population, except for natural variations that may be present to a minor extent. Refers to antibodies obtained from a population that are identical. Monoclonal antibodies are highly specific for a single antigen binding site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

With respect to polynucleotides and polypeptides, “identity” means a comparison that is optionally calculated using the algorithms shown in (1) and (2) below:
(1) Polynucleotide identity is calculated by multiplying the total number of nucleotides in a given sequence by an integer that defines the percent identity divided by 100, and then subtracting the solution from the total number of nucleotides in the sequence. The That is,
nn ≦ xn− (xn · y)
[Where nn is the number of nucleotide changes, xn is the total number of nucleotides in a given sequence, and y is 0.95 for 95%, 0.97 for 97% or 1.7 for 100%. 00 is the symbol for the multiplication operator and solutions that are not integers of xn and y are rounded down to the nearest integer before subtracting from xn]. Changes in the polynucleotide sequence encoding the polypeptide can result in nonsense, missense or frameshift mutations in the coding sequence, thereby changing the polypeptide encoded by the polynucleotide after the change.

(2) Polypeptide identity is calculated by multiplying the total number of amino acids by an integer that defines the percent identity divided by 100, and then subtracting the solution from the total number of amino acids. That is,
na ≦ xa− (xa · y)
[Wherein na is the number of amino acid changes, xa is the total number of amino acids in the sequence, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%. Yes, is the symbol for the multiplication operator, and solutions that are not integers of xa and y are rounded down to the nearest integer before subtracting from xa].

  As used herein, the term “variant (s)” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. In general, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar throughout and are identical in many regions. A variant and reference polypeptide can differ in any combination of one or more substitutions, additions, deletions in the amino acid sequence. The substituted or inserted amino acid residue may or may not be encoded by the genetic code. It will be appreciated by those skilled in the art that certain amino acid substitutions are considered “conservative”. Amino acids are divided into groups based on common side chain properties, and substitutions within the group that maintain all or substantially all of the binding affinity of the antibodies of the invention or antigen-binding fragments thereof are Considered a conservative substitution. See the table below.

Side chain member Hydrophobic Met, Ala, Val, Leu, Ile
Neutral hydrophilicity Cys, Ser, Thr
Acid Asp, Glu
Basic Asn, Gln, His, Lys, Arg
Residues affecting chain orientation Gly, Pro
Aromatic Trp, Tyr, Phe

  In some embodiments of the invention, some, eg, 5-10, 1-5, 1-3, 1-2 amino acid residues, or 1 amino acid residue, in any combination Variants substituted, deleted or added with are included. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-natural variants of polynucleotides and polypeptides can be made by mutagenesis methods, by direct synthesis, and by other recombinant methods known in the art.

  By “isolated” is meant “modified by hand” from its natural state, altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally occurring in a living organism is not “isolated”, but the same polynucleotide or polypeptide separated from its native coexisting material is “isolated” It is. For example, but not limited to, the polynucleotide or polypeptide may be introduced back into the cell even if it is the same species or type of cell from which the polynucleotide or polypeptide was isolated.

  In this specification and the appended claims, the terms “comprising” and “comprising” encompass “consisting of” and “consisting of”. That is, these words are intended to refer to possible inclusions of other elements or numbers not specifically described where the circumstances permit.

  The term “glycosylation profile” as used herein refers to the level of glycosylation in an antibody population.

  In connection with the antibodies of the invention and antigen-binding fragments thereof, as used throughout this specification, the term “specifically binds” refers to antibodies that bind human IGF-1R (hIGF-1R), but others The term “human protein” means not bound or significantly bound. However, this term does not exclude that the antibodies of the present invention may also cross-react with other forms of IGF-1R, such as primate IGF-1R.

  The term “neutralize” as used throughout this specification in connection with the antibodies of the invention and antigen-binding fragments thereof is the biological activity of IGF-1R in the presence of the antibodies of the invention and antigen-binding fragments thereof. Means reduced compared to IGF-1R activity in the absence of such antibodies and antigen binding fragments thereof. Neutralization can result from, but is not limited to, one or more of blocking ligand binding, blocking receptor activation of the ligand, downregulating IGF-1R, or affecting effector function. The level of neutralization is measured in several ways, for example in the LISN cell proliferation assay, which can be performed as described in Example 23 by way of example, using the assay described in the examples below. can do. In this assay, neutralization of IGF-1R is measured by assessing the reduction in tumor cell proliferation in the presence of neutralizing antibodies.

  The level of neutralization can also be measured, for example, in a receptor phosphorylation assay that can be performed as described in Example 13, for example. In this assay, neutralization of IGF-1R is measured by assessing inhibition of receptor phosphorylation in the presence of neutralizing antibodies.

If the antibody or antigen-binding fragment thereof has neutralizing ability, this indicates inhibition of the interaction between the human IGF-1R binding protein (eg hIGF-I or hIGF-II) and its receptor. Antibodies considered to have neutralizing activity against human IGF-1R are less than 10 μg / ml or less than 5 μg / ml in the LISN cell proliferation assay or receptor phosphorylation assay described in Example 23 and Example 13, respectively. Alternatively, it appears to have an IC ₅₀ of less than 2 μg / ml, or less than 1 μg / ml.

  In another aspect of the invention, the antibodies exemplified herein, eg, H0L0 and H0L0 IgG1m (AA) in the LISN cell proliferation assay or receptor phosphorylation assay described in Example 23 and Example 13, respectively. And an antibody or antigen-binding fragment thereof having a neutralizing activity equivalent to that of an antibody retaining the neutralizing activity of H1L0 and H10L0 IgG1m (AA).

  Throughout this specification, amino acid residues in antibody sequences are numbered according to the Kabat format. Similarly, the terms “CDR”, “CDR L1”, “CDR L2”, “CDR L3”, “CDR H1”, “CDR H2”, “CDR H3” are Kabat et al; Sequences of proteins of Immunological Interest Follow the Kabat numbering scheme described in NIH, 1987. As will be apparent to those skilled in the art, there are other definitions of CDR sequences, such as those described in Chothia et al. (1989).

  As will be apparent to those skilled in the art, the term “derived from” not only reveals the origin in the sense that it is the physical origin of the substance, but also structurally (with respect to the primary amino acid sequence) It is intended to indicate substances that are identical but not derived from their reference origin. Therefore, “residues present in the donor antibody from which CDR H3 is derived” does not necessarily have to be purified from the donor antibody.

  The term “stability” as used throughout this specification with respect to the antibodies of the invention and antigen-binding fragments thereof is the value at which the activity of the antibody or antigen-binding fragment, as measured by direct binding ELISA, is at the start of EC-50. And equivalent after 12 days of incubation in serum at −20 ° C., 4 ° C. or 37 ° C.

  “Chimeric antibody” refers to a type of engineered antibody comprising a natural variable domain (light chain and heavy chain) derived from a donor antibody and an acceptor antibody derived light chain and heavy chain constant region associated therewith. Point to.

  A “humanized antibody” is a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin and the remaining immunoglobulin-derived portion of the molecule is derived from one (or more) human immunoglobulin. Point to. In addition, framework support residues can be modified to preserve binding affinity (eg, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989); Hodgson et al., Bio / Technology, 9: 421 (1991)). Suitable human acceptor antibodies may be those selected from conventional databases such as the KABAT® database, the Los Alamos database, and the Swiss Protein database by homology with the nucleotide and amino acid sequences of the donor antibody. . Human antibodies characterized by homology (on an amino acid basis) with the framework region of the donor antibody are suitable for providing a heavy chain constant region and / or a heavy chain variable framework region for insertion of a donor CDR It is. Suitable acceptor antibodies that can provide light chain constant or variable framework regions can be selected in a similar manner. Note that the acceptor antibody heavy and light chains need not originate from the same acceptor antibody. The prior art describes several methods for making such humanized antibodies. See for example EP-A-0239400 and EP-A-054951.

  The term “donor antibody” refers to a first immunoglobulin partner so that a modified immunoglobulin coding region is obtained and expression of a modified antibody with antigen specificity and neutralizing activity characteristic of the donor antibody occurs. Refers to an antibody (monoclonal and / or recombinant) that provides the amino acid sequence of its variable domain, CDR, or functional fragment or analog thereof.

  The term “acceptor antibody” refers to the first immunoglobulin partner with all of the amino acid sequences encoding its heavy and / or light chain framework regions and / or its heavy and / or light chain constant regions (or A donor antibody that provides any but preferably all) refers to a heterologous antibody (monoclonal and / or recombinant). A human antibody is an acceptor antibody.

  “CDR” is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable domain of immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs and three light chain CDRs (ie, CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, “CDR” means all three heavy chain CDRs, or all three light chain CDRs (or all heavy chain CDRs and all light chain CDRs, as appropriate). Antibody structure and protein fold means that other residues are considered to be antigen-binding regions, as is understood by those skilled in the art, for example Chothia et al., ( 1989) Conformations of immunoglobulin hypervariable domains; Nature 342, p877-883.

  CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. The subject CDRs of the present invention are derived from donor antibody variable heavy and light chain sequences and include analogs of native CDRs, which analogs also have the same antigen binding specificity as the donor antibody from which they are derived. And / or have or retain neutralizing ability.

The terms “V _H ” and “V _L ” are used herein to refer to the heavy and light chain variable domains of an antibody, respectively.

  As used herein, the term “effector function” refers to responses mediated by antibody-dependent cytotoxic activity (ADCC) and complement-dependent cytotoxic activity (CDC), Fc-mediated phagocytosis, and FcRn receptor. Is meant to refer to one or more of the recycling of antibodies. Interaction of the antibody constant region with various Fc receptors (FcR) is believed to mediate the effector functions of the antibody. Significant biological effects include effector function, particularly antibody-dependent cytotoxic activity (ADCC), complement consolidation (complement-dependent cytotoxic activity or CDC), phagocytosis (antibody-dependent cell-mediated phagocytosis, That is, ADCP), and antibody half-life / clearance results. In general, the ability to mediate effector functions requires the binding of antibodies to antigens, and not all antibodies mediate their respective effector functions.

  Effector function can be measured in several ways, for example by measuring FCCγRIII binding to natural killer cells or by binding FcγRI to monocytes / macrophages. For example, an antibody or antigen-binding fragment of the invention has increased ADCC effector function as measured against an equivalent wild-type antibody or antigen-binding fragment thereof in a natural killer cell assay. Examples of such assays are Shields et al, 2001 The Journal of Biological Chemistry, Vol. 276, p6591-6604; Chappel et al, 1993 The Journal of Biological Chemistry, Vol 268, p25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.

  Examples of assays for measuring CDC function include those described in 1995 J Imm Meth 184: 29-38.

  Various modifications (modifications) to the heavy chain constant region of the antibody have been made depending on the desired effector properties. Human constant regions that are essentially deficient in the functions of a) activation of complement by the classical pathway and b) mediation of antibody-dependent cytotoxic activity include IgG4 and IgG2 constant regions. IgG1 constant regions containing specific mutations have been separately described to reduce binding to Fc receptors and thus reduce ADCC and CDC (Duncan et al. Nature 1988, 332; 563-564 Lund et al. J. Immunol. 1991, 147; 2657-2662; Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol. 1992, 51; 1-84; Morgan et al. , Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75 (24); 12161-12168). Human IgG1 constant regions containing specific mutations or modified glycosylation at residue Asn297 have also been described to enhance binding to Fc receptors. They have also been shown to enhance ADCC and CDC in some cases (Lazar et al. PNAS 2006, 103; 4005-4010; Shields et al. J Biol Chem 2001, 276; 6591-6604; Nechansky et al. Mol Immunol, 2007, 44; 1815-1817).

  For IgG antibodies, effector functions such as ADCC and ADCP are mediated by the interaction of the heavy chain constant region with the Fcγ receptor family present on the surface of immune cells. In humans, these include FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). A range of effects, such as cytotoxic activity, immune cell activation, phagocytosis, and release of inflammatory cytokines, are induced by the interaction of the antibody bound to the antigen and the formation of the Fc / Fcγ complex. Certain substitutions in the constant region (including S239D / I332E) are known to increase the affinity of the heavy chain constant region for certain Fc receptors and thus enhance the effector function of the antibody (Lazar et al. PNAS 2006).

1. Antibody structure
1.1 Intact Antibodies Intact antibodies include heteromultimeric glycoproteins comprising at least two heavy chains and two light chains. With the exception of IgM, intact antibodies are usually about 150 kDa heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to the heavy chain by one disulfide covalent bond, but the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has an intrachain disulfide bridge. Each heavy chain has one variable domain (V _H ) at one end followed by several constant regions (CH ₁ , CH ₂ , CH ₃ ). Each light chain has one variable region (L) and one constant region at the other end; the heavy chain constant region of the light chain is aligned with the first constant region of the heavy chain, and the light chain variable domain is the heavy chain Alongside the variable domain. The light chain of antibodies from most vertebrate species can be attributed to one of two types, designated κ and λ, based on the amino acid sequence of the constant region. Depending on the amino acid sequence of the heavy chain constant region of their heavy chains, human antibodies can be assigned to five different classes, namely IgA, IgD, IgE, IgG and IgM. IgG and IgA can be further subdivided into subclasses IgG1, IgG2, IgG3 and IgG4; and IgA1 and IgA2. Mutations by animal species exist, and mice and rats have at least IgG2a, IgG2b. The variable domain of an antibody has a particular region that confers binding specificity on the antibody and exhibits special variability called the complementarity determining region (CDR). The relatively conserved portion of the variable domain is called the framework region (FR). Each variable region of the intact heavy and light chains contains 4 FRs joined by 3 CDRs. The CDRs of each chain are held in close proximity by the FR region, and together with the CDRs of the other chain, result in the formation of an antibody antigen binding site. The constant region is not directly involved in antibody binding to antigen, but is involved in antibody-dependent cytotoxic activity (ADCC), phagocytosis through binding to Fcγ receptor, neonatal Fc Various effector functions are shown, including participation in the half-life / clearance rate by the receptor (FcRn), and in complement-dependent cytotoxicity through the C1q component of the complement cascade.

1.1.2 Human antibodies Human antibodies can be made by several methods known to those skilled in the art. Human antibodies can be produced by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines. See Kozbor, J. Immunol 133, 3001, (1984), and Brodeur, Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize the human variable domain repertoire (Winter G, (1994), Annu. Rev. Immunol 12,433-455, Green LL (1999 ), J. Immunol. Methods 231, 11-23).

Several transgenic mouse strains in which the mouse immunoglobulin loci have been replaced with human immunoglobulin gene segments are currently available (Tomizuka K, (2000) PNAS 97,722-727; Fishwild DM (1996) Nature Biotechnol. 14,845-851, Mendez MJ, 1997, Nature Genetics, 15,146-156). After challenge with the antigen, the mouse can produce a repertoire of human antibodies from which the antibody of interest can be selected. Of particular note is the Trimera ^™ system (see Eren R et al. (1998) Immunology 93: 154-161), which transplants human lymphocytes into irradiated mice, human (or other species) lymphocytes, Selected Lymphocyte Antibody System (see SLAM, Babcook et al., PNAS (1996) 93: 7843-7848) which undergoes a large pool of in vitro antibody production procedures followed by deconvolution, limiting dilution and selection procedures , as well as XenomouseII ^TM (Abgenix Inc). Another approach uses Morphodoma ^™ technology available from Morphotek Ink.

  Phage display technology can be used to make human antibodies (and fragments thereof). See McCafferty, Nature, 348, 552-553 (1990) and Griffiths AD et al. (1994) EMBO 13: 3245-3260. With this technique, antibody variable domain genes are cloned in-frame into the major or minor coat protein genes of filamentous bacteriophages such as M13 or fd and displayed as functional antigen-binding fragments on the surface of phage particles. (Usually needs help from helper phage). As a result of selection based on the functional properties of the antibody, a gene encoding an antibody exhibiting such properties is selected. Phage display methods can be used to select antigen-specific antibodies from libraries made from human B cells taken from individuals suffering from a disease or disorder, or from unimmunized human donors (Marks; J. Mol. Bio. 222, 581-597, 1991). If an intact human antibody containing a constant domain is desired, it is necessary to reclon the phage display-derived fragment into a mammalian expression vector containing the desired constant region to establish a stable expression cell line. .

  Affinity maturation (Marks; Bio / technol 10,779-783 (1992)) techniques can be used to improve binding affinity, in which case heavy and light chain variable domains are naturally occurring variants. By sequentially substituting with and selecting based on the improvement in binding affinity, the affinity of the human primary antibody is improved. Variations of the above techniques such as “epitope imprinting” are now available. See WO 93/06213. See also Waterhouse; Nucl. Acids Res 21, 2265-2266 (1993).

1.2 Chimeric and humanized antibodies The use of intact non-human antibodies in the treatment of human diseases or disorders has the potential to cause currently well-proven immunogenicity problems, i. The immune system may recognize intact non-human antibodies as non-self and initiate a neutralizing response. This is particularly evident with multiple doses of non-human antibodies to human patients. Various techniques have been developed over the years to overcome these problems, but these techniques generally provide a relative ease of obtaining non-human antibodies from immunized animals such as mice, rats or rabbits. While maintaining, it is necessary to reduce the proportion of non-human amino acid sequences in intact antibodies. In general, two approaches have been used to accomplish this. The first is a chimeric antibody, which generally comprises a non-human (eg, a rodent such as a mouse) variable domain fused to a human constant region. Since the antigen-binding site of the antibody is localized within the variable domain, the chimeric antibody retains its binding affinity for the antigen, but has acquired the effector function of the human constant region, and thus has the effector function as described above. Can fulfill. Chimeric antibodies are typically made using recombinant DNA methods. DNA encoding the antibody (eg, cDNA) using conventional methods (eg, by using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of the antibody of the invention) Is isolated and sequenced. Hybridoma cells are a typical source of such DNA. After isolation, the DNA is placed in an expression vector, which is then transfected into a host cell such as E. coli, COS cells, CHO cells or myeloma cells that do not inherently produce immunoglobulin proteins to synthesize antibodies. The DNA can be modified by replacing the corresponding sequences of non-human (eg, mouse) H and L constant regions with human L and H chain coding sequences. See, for example, Morrison; PNAS 81, 6851 (1984).

  The second approach relates to the production of humanized antibodies that reduce the non-human content of the antibody by humanizing variable domains. Two techniques for humanization are widely supported. The first is humanization by CDR grafting. The CDR makes a loop near the N-terminus of the antibody, where it forms a surface fitted into the scaffold provided by the framework regions. The antigen binding specificity of an antibody depends primarily on the topography and chemical nature of the CDR surface of the antibody. These characteristics are then determined by the individual CDR conformation, by the relative configuration of the CDRs, and by the nature and configuration of the side chains of the residues that make up the CDRs. A significant reduction in immunogenicity transplants (grafts) only the CDRs of a non-human (eg mouse) antibody (“donor” antibody) onto the human framework (“acceptor framework”) and constant regions. (See Jones et al. (1986) Nature 321,522-525 and Verhoeyen M et al. (1988) Science 239, 1534-1536). However, CDR grafting may not inherently result in complete retention of antigen binding properties, and donor antibody framework residues ("reversion" may be used if significant antigen binding affinity is to be restored. It has often been found that some of them (sometimes called "mutations") need to be stored in humanized molecules (Queen C et al. (1989) PNAS 86, 10,029-10,033, Co, M et al. (1991) Nature 351, 501-502). In this case, to prepare a human framework (FR), a human variable domain that exhibits the greatest sequence homology to a non-human donor antibody is selected from the database. Human FRs can be selected from either human consensus or individual human antibodies. If necessary, key residues from the donor antibody are substituted into the human acceptor framework to preserve the CDR conformation. Although computer modeling of antibodies can be used, it helps to identify such structurally important residues. See WO99 / 48523.

  Alternatively, humanization can be performed by a “veneering” process. Statistical analysis of specific human and mouse immunoglobulin heavy and light chain variable domains reveals that the exact pattern of exposed residues differs between human and mouse antibodies, and that most individual surface positions are a few different It has been shown that residues are strongly preferred (Padlan EA et al. (1991) Mol. Immunol. 28, 489-498, and Pedersen JT et al. (1994) J. Mol. Biol. 235; 959- See 973). Accordingly, non-human Fv immunogenicity can be reduced by replacing exposed residues in the framework region that are different from those normally present in human antibodies. Since the antigenicity of a protein can correlate with accessibility to the surface, replacing surface residues may be sufficient to make the mouse variable domain “invisible” to the human immune system (See also Mark GE et al., (1994), Handbook of Experimental Pharmacology, No. 113: The pharmacology of monoclonal antibodies, Springer-Verlag, pages 105-134) . Such a humanization procedure is called “veneering” because it modifies only the surface of the antibody and leaves the supporting residues intact.

1.3 Bispecific Antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Methods for making such antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, the two heavy chains having different binding specificities. See Millstein et al., Nature 305 537-539 (1983), WO 93/08829 and Traunecker et al., EMBO, 10, 1991, 3655-3659. Due to the random combination of H and L chains, a mixture of 10 different antibody structures can occur, only one of which has the desired binding specificity. Another approach involves fusing a variable domain with the desired binding specificity to a heavy chain constant region comprising at least a portion of the hinge region, the CH2 and CH3 regions. In one embodiment, the CH1 region containing the site necessary for binding to the light chain is present in at least one of the fusions. These fusions and, if necessary, the DNA encoding the light chain are inserted into separate expression vectors and then co-transfected into a suitable host organism. It is also possible to insert the coding sequence of two strands or all three strands into one expression vector. In one approach, a bispecific antibody is composed of an H chain having a first binding specificity in one arm and an HL chain pair that provides a second binding specificity in the other arm. See WO94 / 04690. See also Suresh et al., Methods in Enzymology 121, 210, 1986.

  In certain embodiments of the invention, a bispecific antibody is provided, wherein the binding specificity of at least one of the antibodies is to hIGF-1R, said antibody neutralizing the activity of hIGF-1R To do. Such antibodies can further comprise a human constant region of an IgG isotype, eg, IgG1, IgG2, IgG3 or IgG4. The antibodies of the invention may also be multispecific, eg multispecific antibodies formed by a collection of several antigen binding fragments.

1.4 Antigen-binding fragments Such antigen-binding fragments are partial heavy or light chain variable sequences that retain the same antigen-binding specificity and the same or similar neutralizing activity as the antibody from which they are derived (eg Small deletions in the amino or carboxy terminus of immunoglobulin variable domains).

In certain embodiments of the invention, antigen binding fragments are provided that neutralize the activity of hIGF-1R. Such fragments are intact and / or humanized and / or functional antigen-binding fragments of chimeric antibodies, such as Fab, Fab ′, F (ab ′) ₂ , Fv, ScFv fragments of the above antibodies. be able to. Traditionally, such fragments are produced by proteolysis of intact antibodies, for example by papain degradation (see eg WO 94/29348), but can also be produced directly from transgenic host cells by genetic recombination. . For the creation of ScFv, see Bird et al. (1988) Science, 242, 423-426. In addition, antigen-binding fragments can be made using various genetic engineering techniques described below.

The Fv fragment appears to have a lower interaction energy between its two chains than the Fab fragment. In order to stabilize the binding of the V _H and V _L domains, these domains are composed of peptides (Bird et al. (1988) Science 242, 423-426, Huston et al. PNAS, 85, 5879-5883), disulfide bridges ( Glockshuber et al. (1990) Biochemistry, 29, 1362-1367) and the “knob in hole” mutation (Zhu et al. (1997), Protein Sci., 6, 781-788). ScFv fragments can be generated by methods well known to those skilled in the art. See Whitlow et al. (1991) Methods companion Methods Enzymol, 2, 97-105 and Huston et al. (1993) Int. Rev. Immunol 10, 195-217. ScFv can be produced in bacterial cells such as E. coli, but is preferably produced in eukaryotic cells. The disadvantages of ScFv are that the product is monovalent, thus making it impossible to increase the binding force due to multivalent binding, and the short half-life. Attempts to overcome these problems include divalent (ScFv ′) ₂ , which can be obtained from a ScFv containing an additional C-terminal cysteine by chemical coupling (Adams et al. (1993) Can. Res 53, 4026-4034, and McCartney et al. (1995) Protein Eng. 8, 301-314), or by spontaneous site-specific dimerization of ScFv containing unpaired C-terminal cysteine residues (Kipriyanov Et al. (1995) Cell. Biophys 26, 187-204).

Alternatively, a multimer can be formed in ScFv by shortening the peptide linker to 3-12 residues to form a “diabody”. See Holliger et al., PNAS (1993), 90, 6444-6448. By further reducing the linker, ScFv trimers ("Triabodies", see Kortt et al. (1997) Protein Eng, 10, 423-433) and tetramers ("Tetrabodies", Le Gall et al. (1999) FEBS Lett, 453, 164-168). The construction of bivalent ScFv molecules is described in “miniantibodies” (see Pack et al. (1992) Biochemistry 31, 1579-1584) and “minibodies” (Hu et al. (1996), Cancer Res. 56. , 3055-3061) can also be achieved by gene fusion with a protein dimerization motif that can form. ScFv-ScFv tandems ((ScFv) ₂ ) can also be made by linking two ScFv units with a third peptide linker. See Kurucz et al. (1995) J. Immol. 154, 4576-4582. Bispecific diabodies can be made by non-covalent attachment of two single-chain fusion products consisting of a _VH domain from another antibody linked to the _VL domain of one antibody by a short linker. . See Kipriyanov et al. (1998), Int. J. Can 77, 763-772. The stability of such a bispecific diabody can be achieved by introducing a disulfide bridge, or the above “knob in hole” mutation, or by connecting two hybrid ScFv fragments via a peptide linker. It can be enhanced by forming a chain diabody (ScDb). See Kontermann et al. (1999) J. Immunol. Methods 226 179-188. A tetravalent bispecific molecule is obtained, for example, by fusing a ScFv fragment to the CH3 domain of an IgG molecule or to a Fab fragment via the hinge region. Co
See loma et al. (1997) Nature Biotechnol. 15, 159-163. Alternatively, tetravalent bispecific molecules have been made by fusion of bispecific single stranded diabodies (see Alt et al. (1999) FEBS Lett 454, 90-94). Smaller tetravalent bispecific molecules dimerize ScFv-ScFv tandems with helix-loop-helix motif-containing linkers (see DiBi miniantibodies, Muller et al. (1998) FEBS Lett 432, 45-49) Or dimerization of single chain molecules containing four antibody variable domains (V _H and V _L ) in an orientation that prevents intramolecular pairing (tandem diabodies, Kipriyanov et al., (1999) J. Mol. Biol. 293, 41-56)). Bispecific F (ab ′) ₂ fragments can be generated by chemical coupling of Fab ′ fragments or by heterodimerization with leucine zippers (Shalaby et al. (1992) J. Exp. Med 175, 217-225, and Kostelny et al. (1992), J. Immunol. 148, 1547-1553).

The expression “immunoglobulin single variable domain” means an antibody variable domain (V _H , V _HH , V _L ) that specifically binds an antigen or epitope independently of a different V region or domain. An immunoglobulin single variable domain is one in which a single immunoglobulin variable domain does not require another different variable region or domain for antigen binding (ie, an immunoglobulin single variable domain is independent of another variable domain). When bound to an antigen), it can exist in the form of other different variable regions or variable domains (eg, homo- or heteromultimers).

Isolated _VH and _VL domains (Domantis plc) can also be used. See U.S. Patent Nos. 6,248,516; 6,291,158; 6,172,197. These are known as domain antibodies. A “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” (this term is used herein) capable of binding to an antigen. Immunoglobulin single variable domains may be human antibody variable domains, but single antibody variable domains derived from other species such as rodents (eg, disclosed in WO 00/29004, shark shark and camelids ( Camelid) V _HH dAb, etc.). Camelid V _HH is an immunoglobulin single variable domain polypeptide derived from species such as camels, llamas, alpaca, dromedaries and guanacos that produce heavy chain antibodies that naturally lack light chains. Such V _HH domains can be humanized by standard techniques available in the art, and such domains are also considered “domain antibodies” in the present invention. As used herein, “V _H ” includes camelid V _HH domains.

In certain embodiments, an antigen-binding fragment that specifically binds to hIGF-1R and neutralizes the activity of hIGF-1R (eg, ScFv, Fab, Fab ′, F (ab ′) ₂ ), or as described above Are provided with genetically engineered antigen-binding fragments. Antigen binding fragments are CDR H3 shown in SEQ ID NO: 1, CDR H2 shown in SEQ ID NO: 2, CDR H1 shown in SEQ ID NO: 3, CDR L1 shown in SEQ ID NO: 4, CDR L2 shown in SEQ ID NO: 5, And one or more of the CDR L3 sequences shown in SEQ ID NO: 6.

1.5 Heteroconjugate antibodies Heteroconjugate antibodies also form an embodiment of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies formed using any convenient cross-linking method. See, for example, US Pat. No. 4,676,980.

  Furthermore, the present invention includes combinations of antibodies and antigen-binding fragments, eg, ScFv combined with one or more domain antibodies and / or monoclonal antibodies.

1.6 Interaction of constant regions of other modified antibodies with various Fc receptors (FcγRs) is thought to mediate the effector functions of antibodies, which include antibody-dependent cytotoxic activity ( ADCC), complement binding, phagocytosis, and antibody half-life / clearance. Depending on the desired properties, various modifications of the constant regions of the antibodies of the invention can be made. For example, specific mutations within the constant region that render an otherwise soluble antibody insoluble are detailed in EP0629 240B1 and EP0307 434B2, or the antibody incorporates a salvage receptor binding epitope, The blood half-life can be increased. See U.S. Pat. No. 5,739,277. There are five currently known human Fcγ receptors, FcγR (I), FcγRIIa, FcγRIIb, FcγRIIIa and neonatal FcRn. Shields et al. (2001) J. Biol. Chem 276, 6591-6604, where a common set of IgG1 residues is involved in binding to all FcγRs, whereas FcγRII and FcγRIII are outside of this common set. It has been clarified that a separate site is used. A group of IgG1 residues reduced binding to all FcγRs when Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239 were changed to alanine. Both are present in the IgG CH2 domain and form a cluster near the hinge connecting CH1 and CH2. FcγRI uses only a common set of IgG1 residues for binding, whereas FcγRII and FcγRIII interact with distinct residues in addition to the common set. Some residue changes only reduced FcγRII binding (eg, Arg-292) or only FcγRIII binding (eg, Glu-293). One mutant showed enhanced binding to FcγRII or FcγRIII, but did not affect binding to the other receptor (eg, Ser-267Ala enhanced binding to FcγRII, but not FcγRIII). Did not affect the binding). Other mutants also showed enhanced binding to FcγRII or FcγRIII, but decreased binding to the other receptor (eg, Ser-298Ala enhanced binding to FcγRIII but not to FcγRII). Decreased). For FcγRIIIa, the IgG1 variant that binds best had a combination of alanine substitutions at Ser-298, Glu-333 and Lys-334. The neonatal FcRn receptor is thought to be involved in both antibody clearance and transcytosis across tissues (Junghans R. P (1997) Immunol. Res 16. 29-57 and Ghetie et al. (2000) Annu. Rev. Immunol. 18, 739-766). Human IgG1 residues that have been determined to interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. Switching at any of these positions described in this section can allow for increased serum half-life and / or modification of the effector properties of the antibodies of the invention.

  Other modifications include glycosylation (glycosylation) variants of the antibodies of the invention. Glycosylation (glycosylation) at conserved positions within the constant region of an antibody is known to have a significant effect on antibody function, particularly effector functions as described above. See, for example, Boyd et al. (1996), Mol. Immunol. 32, 1311-1318. A glycosylated variant of the antibody of the present invention or an antigen-binding fragment thereof, to which one or more sugar chains have been added, substituted, deleted, or modified, is envisaged. Introduction of an asparagine-X-serine or asparagine-X-threonine motif creates a site where sugar chains can be linked enzymatically, and can be used to manipulate glycosylation of antibodies. In Raju et al. (2001) Biochemistry 40, 8868-8876, terminal sialylation of TNFR-IgG immunoadhesin is achieved by regalactosylation with β-1,4-galactosyltransferase and / or α-2,3-sialyltransferase and Increased by re-sialylation. Increased terminal sialylation is believed to increase the half-life of the immunoglobulin. Antibodies, like most glycoproteins, are typically produced as a mixture of glycoforms. Such a mixture is particularly apparent when antibodies are produced in eukaryotes, especially mammalian cells. Various methods have been developed to produce the identified glycoforms. Zhang et al., Science (2004), 303, 371, Sears et al., Science, (2001) 291, 2344, Wacker et al. (2002) Science, 298 1790, Davis et al. (2002) Chem. Rev. 102, 579, Hang Et al. (2001) Acc. Chem. Res 34, 727. Accordingly, the invention includes a plurality of glycoforms as described herein comprising a specified number (eg, no more than 7, eg, no more than 5, eg 2 or 1) of said antibody or antigen-binding fragment thereof. Assume a (monoclonal) antibody (which can be an IgG1 isotype, eg, IgG1).

  Still other embodiments of the invention include antibodies of the invention or antigen-binding fragments thereof conjugated to non-proteinaceous polymers such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene. Conjugation of proteins with PEG is an established technique for reducing the antigenicity and immunogenicity of proteins while increasing the half-life of the protein. The use of PEGylation with molecules of different molecular weight and shape (linear or branched) has been investigated with intact antibodies, as well as Fab 'fragments. See Koumenis I.L. et al. (2000) Int. J. Pharmaceut. 198: 83-95.

2. Production Method Although the antibody of the present invention can be produced as a polyclonal population, it is more preferred to produce the antibody as a monoclonal population (that is, as a substantially uniform population comprising the same antibody against a specific antigen-binding site). It will be apparent to those skilled in the art that a population means more than one antibody species. Antibodies of the present invention can be found in goats (see Pollock et al. (1999), J. Immunol. Methods 231: 147-157), chickens (Morrow KJJ (2000) Genet. Eng. News 20: 1-55). ), Mouse (Pollock et al.), Or plant (Doran PM, (2000) Curr. Opinion Biotechnol. 11, 199-204, Ma JK-C (1998), Nat. Med. 4; 601-606, Baez J Et al., BioPharm (2000) 13: 50-54; Stoger E et al. (2000) Plant Mol. Biol. 42: 583-590). Antibodies can also be made by chemical synthesis. However, the antibodies of the invention are typically generated using recombinant cell culture techniques well known to those skilled in the art. The polynucleotide encoding the antibody is isolated and inserted into a replicable vector for subsequent cloning (amplification) or expression. Particularly useful when the host cell is CHO or NS0 (see below), one useful expression system is the glutamate synthetase system (such as that sold by Lonza Biologics). Polynucleotide encoding the antibody is readily isolated and sequenced by conventional methods (eg, oligonucleotide probes). Vectors that can be used include plasmids, viruses, phages, transposons, minichromosomes (plasmids are typical examples). In general, such vectors are signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription terminations operably linked to light and / or heavy chain polynucleotides to facilitate expression. It further contains a sequence. Polynucleotides encoding light and heavy chains can be inserted into separate vectors and transfected into the same host cell, or both heavy and light chains can be inserted into the same vector, if desired And can be transfected into a host cell. Thus, according to one aspect of the present invention, there is provided a method for constructing a vector encoding the light chain and / or heavy chain of the antibody of the present invention or an antigen-binding fragment thereof, which method comprises the antibody of the present invention. Inserting a polynucleotide encoding the light chain and / or heavy chain of the vector into a vector.

  It is known to those skilled in the art that a synthetic gene encoding the same protein as a natural or wild type gene can be designed by changing the codons used for that gene.

  These design approaches include the process of replacing a codon in a gene that is rarely used in a mammalian gene with a codon that is more frequently used for that amino acid in the mammalian gene. This process, so-called codon optimization, is intended to increase the total protein level produced by the host cell when transfected with the codon optimized gene compared to the level when transfected with the wild type sequence. Used. Several methods have been published (Nakamura et. Al., Nucleic Acids Research 1996, 24: 214-215; WO 98/34640; WO 97/11086).

  Codon frequency can be derived from literature sources for highly expressed genes of many species (see, eg, Nakamura et al. Nucleic Acids Research 1996, 24: 214-215). A codon usage table for humans has also been published (WO2005025614).

  Due to the redundancy of the genetic code, a polynucleotide that is an alternative to the polynucleotide described herein that encodes a polypeptide of the invention (especially its codons are optimized for expression in a given host cell) Can be readily understood by those skilled in the art.

3.1 Signal Sequence The antibody of the present invention can be prepared as a fusion protein containing a heterologous signal sequence having a specific cleavage site at the N-terminus of the mature protein. The signal sequence should be recognized and processed by the host cell. For prokaryotic host cells, the signal sequence can be, for example, alkaline phosphatase, penicillinase, or thermostable enterotoxin II leader. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader, an alpha factor leader, or an acid phosphatase leader. See WO90 / 13646. In mammalian cell systems, viral secretion leaders such as herpes simplex gD signal, as well as native immunoglobulin signal sequences are preferred. Typically, the signal sequence is linked in reading frame to DNA encoding the antibody of the invention.

3.2 Origin of replication The origin of replication is well known in the art, pBR322 is compatible with most gram-negative bacteria, 2μ plasmid is compatible with most yeasts, and from various viruses such as SV40, Polyoma, adenovirus, VSV or BPV are suitable for most mammalian cells. In general, origin of replication elements are not required for mammalian expression vectors, but SV40 can be used because it contains the early promoter.

3.3 Selectable Markers Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins (eg, ampicillin, neomycin, methotrexate or tetracycline), or (b) auxotrophic Encodes a protein that complements the deficiency of or supplements nutrients not available in complex media. The selection scheme may involve stopping the growth of the host cell. Cells that have been successfully transformed with a gene encoding an antibody of the invention survive, for example, due to drug resistance conferred by a selectable marker. Another example is the so-called DHFR selection marker, in which case the transformants are cultured in the presence of methotrexate. In an exemplary embodiment, cells are cultured with increasing amounts of methotrexate to amplify the copy number of the foreign gene of interest. CHO cells are a particularly useful cell line for DHFR selection. Yet another example is the glutamate synthetase expression system (Lonza Biologics). A selection gene suitable for use in yeast is the trp1 gene. See Stinchcomb et al., Nature 282, 38, 1979.

3.4 Promoters A promoter suitable for expression of an antibody of the invention is operably linked to the DNA / polynucleotide encoding the antibody. Promoters for prokaryotic hosts include the proA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan and hybrid promoters such as Tac. Suitable promoters for expression in yeast cells include 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose There are 6-phosphate isomerase, 3-phosphoglycerate mutase and glucokinase. Inducible yeast promoters include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein, and enzymes responsible for nitrogen metabolism or maltose / galactose utilization. Promoters for expression in mammalian cell lines include viral promoters such as polyoma, fowlpox and adenovirus (eg, adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (specifically the immediate early stage). Gene promoter), retrovirus, hepatitis B virus, actin, Rous sarcoma virus (RSV) promoter, and early or late simian virus 40. Of course, the choice of promoter is based on the appropriate compatibility with the host cell used for expression. Thus, in certain embodiments, a first comprising an RSV and / or SV40 and / or CMV promoter, a DNA encoding a light chain variable domain (V _L ) of the invention, a κC region, together with a neomycin and ampicillin resistance selectable marker. plasmid, and RSV or SV40 promoter, DNA encoding the heavy chain variable domain _{(V H)} of the present invention, DNA encoding the γ1 constant region, a second plasmid containing the DHFR and ampicillin resistance markers are provided.

3.5 Enhancer Element If desired, an enhancer element operably linked to a promoter element in the vector can be used, for example, for expression in higher eukaryotes. Suitable mammalian enhancer sequences include enhancer elements derived from globin, elastase, albumin, fetoprotein and insulin. Alternatively, enhancer elements of eukaryotic virus origin such as SV40 enhancer (bp100-270), cytomegalovirus early promoter enhancer, polymer enhancer, baculovirus enhancer, or mouse IgG2a locus may be used (WO04 / See 009823). An enhancer can be located on the vector at a site upstream of the promoter.

3.5.5 Polyadenylation signal In eukaryotic systems, a polyadenylation signal is operably linked to a DNA / polynucleotide encoding an antibody of the invention. Such signals are typically placed 3 'to the open leasing frame. In mammalian systems, non-limiting examples include signals from growth hormone, elongation factor 1α and viral (eg, SV40) genes, or retroviral long terminal repeats. In yeast systems, non-limiting examples of polyadenylation / termination signals include signals from phosphoglycerate kinase (PGK) and alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, polyadenylation signals are generally not needed, and it is customary to use shorter and clearer terminator sequences instead. Of course, the selection of polyadenylation / termination sequences is based on the appropriate compatibility with the host used for expression.

3.6 vectors encoding antibodies of the host cell present invention cloning or host cells suitable for expression, is a prokaryotic cell, yeast or higher eukaryotic cells. Suitable prokaryotic cells include eubacteria, such as the family Enterobacteriaceae, Escherichia such as E. coli (eg ATCC 31,446; 31,537; 27,325), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescens, and Shigella ) And the like, and B. subtilis and B. subtilis of Bacilli. There are also Streptomyces, such as B. licheniformis (see DD 266 710), and Pseudomonas P. aeruginosa. Among yeast host cells, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces (eg, ATCC 16,045; 12,424; 24178; 56,500), Yarrowia (y) EP 402226), Pichia Pastoris (see also EP183070, Peng et al., J. Biotechnol. 108 (2004) 185-192), Candida, Trichoderma reesia (EP244234), Also contemplated are Penicillin, Tolypocladium and Aspergillus hosts, such as A. nidulans and A. niger.

  Prokaryotic and yeast host cells are specifically contemplated by the present invention, but the host cells of the present invention are higher eukaryotic cells. Suitable higher eukaryotic host cells include mammalian cells such as COS-1 (ATCC number CRL 1650), COS-7 (ATCC CRL 1651), human embryonic kidney cell line 293, pup hamster kidney cells (BHK). ) (ATCC CRL.1632), BHK570 (ATCC number: CRL 10314), 293 (ATCC number CRL 1573), Chinese hamster ovary cell CHO (eg CHO-K1, ATCC number: CCL 61, DHFR-CHO cell line, eg DG44 and others (see Urlaub et al. (1986) Somatic Cell Mol. Genet. 12, 555-556)), particularly CHO cell lines suitable for suspension culture, mouse Sertoli cells, monkey kidney cells, African green monkey kidney cells (ATCC CRL-1587), HELA cells, canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), HepG2 and myeloma or lymphoma cells such as NS0 (US Pat. No. 5,807,715). See), Sp2 / 0, Y0.

  Accordingly, in certain embodiments of the invention, there is provided a stably transformed host cell comprising a vector encoding the heavy and / or light chain of an antibody or antigen-binding fragment thereof described herein. . Such a host cell comprises a first vector encoding a light chain and a second vector encoding the heavy chain.

Bacterial fermentation bacterial systems are particularly suitable for the expression of antigen-binding fragments. Such fragments are localized in the cell or in the periplasm. Insoluble periplasmic proteins can be extracted and refolded to form active proteins according to methods known to those skilled in the art. See Sanchez et al. (1999) J. Biotechnol. 72, 13-20 and Cupit PM et al. (1999) Lett Appl Microbiol, 29, 273-277.

3.7 Cell Culture Method Host cells transformed with a vector encoding the antibody of the present invention or an antigen-binding fragment thereof can be cultured by methods known to those skilled in the art. Host cells can be cultured in spinner flasks, roller bottles, or hollow fiber systems, but for large-scale production, a stirred tank reactor is used, especially for suspension culture. The agitation tank is preferably adapted for aeration using, for example, a sparger, baffle or low shear impeller. For bubble columns and airlift reactors, direct aeration with air or oxygen bubbles can be used. When culturing host cells in serum-free medium, a cytoprotective substance, such as Pluronic F-68, is added to help prevent cells from being damaged as a result of the aeration process. Depending on the characteristics of the host cell, the microcarrier can be used as a growth substrate for an anchorage-dependent cell line, or the cells can be adapted for suspension culture (this is common). The cultivation of host cells, especially invertebrate host cells, can be performed in a variety of ways, including fed-batch culture, repeated batch processing (see Drapeau et al. (1994) cytotechnology 15: 103-109), extended batch or perfusion culture. Mode can be used. Recombinantly transformed mammalian host cells can be cultured in serum-containing media such as fetal calf serum (FCS), such as Keen et al. (1995) Cytotechnology 17: 153. -163, or a commercial growth medium such as ProCHO-CDM or UltraCHO ^™ (Cambrex NJ, USA), if necessary, an energy source such as glucose and a synthetic growth factor such as recombinant insulin. Add and incubate. Serum-free culture of host cells may require that the cells are adapted for growth in serum-free conditions. One adaptation method is to culture these host cells in serum-containing medium and repeatedly replace 80% of the medium with serum-free medium (eg, Scharfenberg) so that the host is adapted to serum-free conditions. K et al. (1995) "Animal Cell technology": Development towards the 21st century (Beuvery EC et al., Ed.), Pages 619-623, Kluwer Academic publishers Wanna)

  The antibodies of the invention secreted into the medium can be recovered and purified using a variety of techniques to provide a degree of purification suitable for the intended application. For example, the use of the antibodies of the present invention for the treatment of human patients generally requires a purity of at least 95%, more typically 98% or 99% or greater (as opposed to crude media). ). In a first example, cell debris is removed from the medium, typically by centrifugation, and then a clarification step is performed, for example using microfiltration, ultrafiltration and / or depth filtration. Various other techniques such as dialysis and gel electrophoresis, and chromatographic methods such as hydroxyapatite (HA), affinity chromatography (optionally including an affinity tag labeling system such as polyhistidine) and / or Alternatively, hydrophobic interaction chromatography (HIC, see US Pat. No. 5,429,746) can be utilized. In certain embodiments, the antibodies of the present invention are captured using protein A or G affinity chromatography after various clarification steps and then further ion exchange and / or HA chromatography, anion or cation exchange. Chromatography steps such as size exclusion chromatography, as well as ammonium sulfate precipitation. In general, various virus removal steps are also performed (eg, nanofiltration using a DV-20 filter or the like). After these various steps, a purified (preferably monoclonal) preparation containing at least 75 mg / ml or more, for example 100 mg / ml or more, of the antibody of the invention or antigen-binding fragment thereof is obtained, and thus the preparation is of the present invention. An embodiment is configured. Suitably, the preparation is substantially free of aggregates of antibodies of the invention.

4). Pharmaceutical Compositions The above-described purified preparations (particularly monoclonal preparations) of the antibodies of the present invention can be incorporated into pharmaceutical compositions for use in the treatment of human diseases and disorders as follows. Rheumatoid arthritis, psoriasis, or cancer such as acute lymphoblastic leukemia, adrenocortical cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, colorectal cancer, basal Cell carcinoma, extrahepatic cholangiocarcinoma, bladder cancer, osteosarcoma / malignant fibrous histiocytoma bone cancer, brain tumor (eg brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymocytes , Medulloblastoma, tent undifferentiated neuroectodermal tumor, visual tract and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, unknown primary cancer, primary Central nervous system, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer ,leather T cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma eye cancer, retinoblastoma Eye cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor (eg extracranial, extragonadal and ovary), gestational trophoblastic tumor, glioma (eg , Adult, pediatric brainstem, pediatric cerebral astrocytoma, pediatric visual tract and hypothalamus), hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamus and visual tract Glioma, intraocular melanoma, pancreatic islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney (renal cell) cancer, laryngeal cancer, leukemia (eg, acute lymphoblastic, acute myeloid, chronic lymphocytic, Chronic myeloid and hair cells), lip and oral cancer, liver cancer Non-small cell lung cancer, small cell lung cancer, lymphoma (eg AIDS-related, Burkitt, skin T cells, Hodgkin, non-Hodgkin, and primary central nervous system), Waldenstrom macroglobulinemia, bone malignant fibrosis Histiocytoma / osteosarcoma, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cervical cancer of unknown primary, multiple endocrine tumor syndrome, multiple bone marrow Tumor / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia, chronic myelogenous leukemia, multiple myeloma, chronic myeloproliferative disorder, nasal cavity and sinus Cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer , Islet cell pancreatic cancer, sinus and nasal cavity cancer Parathyroid cancer, penile cancer, pheochromocytoma, pineoblastoma, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, kidney Cellular (renal) cancer, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, Merkel cell skin carcinoma, small intestine cancer , Soft tissue sarcoma, squamous cell carcinoma, cutaneous T cell lymphoma, testicular cancer, thymoma, thymic cancer, thyroid cancer, gestational trophoblastic tumor, carcinoma of unknown primary site, cancer of unknown primary site, urethral cancer, endometrial uterine cancer Uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

  Typically, such compositions comprise a pharmaceutically acceptable carrier that is known and required by acceptable pharmaceutical practice. See, for example, Remingtons Pharmaceutical Sciences, 16th edition, (1980), Mack Publishing Co. Examples of such carriers are sterile carriers such as saline, Ringer's solution or dextrose solution buffered with a suitable buffer in the range of pH 5-8. A pharmaceutical composition for injection (eg, by intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular or intraportal administration) or continuous infusion is suitable in the absence of visible particulate matter. 1 ng to 100 mg of antibody, for example 5 mg to 25 mg of antibody can be contained. Methods for preparing such pharmaceutical compositions are well known to those skilled in the art. In certain embodiments, the pharmaceutical composition comprises 0.1 ng to 100 mg of an antibody of the invention in a unit dosage form, optionally with instructions. The pharmaceutical compositions of the present invention may be lyophilized so that they can be reconstituted prior to administration according to methods well known or apparent to those skilled in the art. Where an embodiment of the invention includes an antibody of the invention having an IgG1 isotype, a chelate (eg, sodium citrate) or a copper chelator such as EDTA or histidine is added to the pharmaceutical composition to produce the antibody The degree of decomposition via copper can be reduced. See EP0612251.

  Effective dosages and treatment regimens for administering the antibodies of the invention or antigen-binding fragments thereof are generally determined empirically and depend on factors such as the age, weight and health status of the patient and the disease or disorder to be treated. Is done. These factors are within the discretion of the attending physician. Guidance in selecting appropriate doses can be found, for example, in Smith et al. (1977) "Antibodies in human diagnosis and therapy", Raven Press, New York, but generally 1 mg to 1000 mg.

  Conveniently, a pharmaceutical composition comprising instructions for use and comprising a kit of parts of the antibody or antigen-binding fragment thereof of the present invention together with another agent is also envisaged by the present invention.

  The present invention further provides a therapeutically effective amount of a therapeutically effective amount for use in the treatment of a disease responsive to neutralization of the interaction between IGF-I and IGF-1R or between IGF-II and IGF-1R. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein is envisioned.

In the present invention, a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal humanized antibody is provided, wherein the antibody comprises a V _H domain selected from the group consisting of SEQ ID NO: 14, and a group consisting of SEQ ID NO: 16. A _VL domain selected from

In the present invention, a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal humanized antibody is provided, wherein the antibody comprises a V _H domain selected from the group consisting of SEQ ID NO: 15, and a group consisting of SEQ ID NO: 16. A _VL domain selected from

  Conveniently, a pharmaceutical composition comprising a kit of parts of the antibody or antigen-binding fragment thereof of the present invention together with such another agent, together with instructions for use depending on the circumstances, is also used in the present invention. is assumed.

  The present invention further comprises a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof as described herein for use in the treatment of a disease responsive to neutralization of IGF-1R activity. Is assumed.

  In another embodiment of the invention, the pharmaceutical composition comprises human diseases and disorders, such as: rheumatoid arthritis, psoriasis, or cancer, such as acute lymphoblastic leukemia, adrenocortical cancer, AIDS-related cancer, AIDS-related lymphoma, Anal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, colorectal cancer, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, osteosarcoma / malignant fibrous histiocytoma bone cancer, brain tumor (eg, brain stem nerve Glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratent undifferentiated neuroectodermal tumor, visual tract and hypothalamic glioma), breast cancer, bronchi Adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, unknown primary cancer, primary central nervous system, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancer, chronic lymphocyte Sex white blood Chronic myelogenous leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer, cutaneous T cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family sarcoma, extracranial germ cell tumor, extragonadal embryo Cellular tumors, extrahepatic cholangiocarcinoma, intraocular melanoma eye cancer, retinoblastoma eye cancer, gallbladder cancer, gastric (Stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor (eg, skull) , Extragonadal, and ovary), gestational trophoblastic tumor, glioma (eg, adult, childhood brain stem, childhood cerebral astrocytoma, childhood visual tract, and hypothalamus), hair cell leukemia, head and neck cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamus and visual tract glioma, intraocular melanoma, islet cell carcinoma (pancreatic endocrine), Kaposi sarcoma, kidney (renal cell) cancer, laryngeal cancer Leukemia (eg, acute lymphoblastic, acute myeloid, chronic Lymphoid, chronic myeloid and hair cells), lip and oral cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma (eg AIDS related, Burkitt, skin T cells, Hodgkin, non-Hodgkin, And primary central nervous system), Waldenstrom macroglobulinemia, bone malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelial Tumor, metastatic squamous cervical cancer of unknown primary, multiple endocrine tumor syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia Chronic myelogenous leukemia, multiple myeloma, chronic myeloproliferative disease, nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / bone malignant fibrous histiocytoma, Ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor Ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineoblastoma, pituitary tumor, plasma cell tumor / multiple myeloma , Pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, soft tissue sarcoma Uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cutaneous T cell lymphoma, testicular cancer, thymoma, thymic cancer, thyroid cancer, gestational trophoblast Tumor, carcinoma of unknown primary site, cancer of unknown primary site, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Used to treat Wilms tumor In combination with other therapeutic agents or radiation therapy, for example, other classes of drugs included: mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibition Agents, enzymes, topoisomerase inhibitors, anti-survival factors, biological response modifiers, anti-hormones, anti-angiogenic agents such as anti-growth factor receptor antagonists such as trastuzumab (Herceptin), Erbitux (cetuximab), anti-growth factor antibodies such as Chemotherapy such as bevacizumab (Avastin), platelet derived growth factor receptor (PDGFR), nerve growth factor (NGFR), antagonist of fibroblast growth factor receptor (FGFR), small molecule tyrosine kinase inhibitors such as lapatinib, gefitinib Agents such as gemcitabine, irinotecan, paclitaxel, shi Suplatin, doxorubicin, topotecan, cyclophosphamide, melphalan, dacarbazine, daunorubicin, aminocamptothecin, etoposide, teniposide, adriamycin, 5-fluorouracil, cytosine arabinoside (Ara-C), thiotepa, taxotere, busulfan, cytoxin, Antibodies are included in combination with taxol, methotrexate, vinblastine, bleomycin, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, carminomycin, aminopterin, dactinomycin.

  The antibodies or antigen-binding fragments thereof of the present invention can be used in combination with one or more other therapeutically active agents or radiation, eg, in combination with other classes of drugs included below: mitotic inhibitors, alkylation Agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival factors, biological response modifiers, anti-hormones, anti-angiogenic agents such as anti-growth factor receptors Antagonists such as trastuzumab (Herceptin), Erbitux (cetuximab), anti-growth factor antibodies such as bevacizumab (Avastin), platelet derived growth factor receptor (PDGFR), nerve growth factor (NGFR), fibroblast growth factor receptor (FGFR) ) Antagonists, small molecule anti-IGF-1R agents, small molecule tyrosine kinases Pesticides such as lapatinib, gefitinib, and chemotherapeutic agents such as gemcitabine, irinotecan, paclitaxel, cisplatin, doxorubicin, topotecan, cyclophosphamide, melphalan, dacarbazine, daunorubicin, aminocamptothecin, etoposide, tenaposide, adriamycin, 5-fluorou , Cytosine arabinoside (Ara-C), thiotepa, taxotere, busulfan, cytoxin, taxol, methotrexate, vinblastine, bleomycin, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, carminomycin, aminopterin, dac Tinomycin.

  Accordingly, in a further embodiment, the present invention relates to the use of such combinations in the treatment of diseases where IGF-1 receptor signaling contributes to the disease or where neutralization of receptor activity is beneficial, as well as Provided is the use of an antibody or antigen-binding fragment thereof in the manufacture of a medicament for combination therapy of disorders (combination therapy): rheumatoid arthritis, psoriasis, or cancer such as acute lymphoblastic leukemia, adrenocortical cancer, AIDS-related cancer , AIDS-related lymphoma, anal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, colorectal cancer, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, osteosarcoma / malignant fibrous histiocytoma bone cancer, brain tumor ( For example, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, tent anaplastic neuroectodermal tumor, visual Tract and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, unknown primary cancer, primary central nervous system, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma Tumor, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, intraocular melanoma eye cancer, retinoblastoma eye cancer, gallbladder cancer, gastric (Stomach) Cancer, gastrointestinal carcinoid tumor, germ cell tumor (eg extracranial, extragonadal, and ovary), gestational trophoblastic tumor, glioma (eg adult, pediatric brainstem, pediatric cerebral astrocytoma, pediatric visual tract, And hypothalamus), Hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamus and visual tract glioma, intraocular melanoma, islet cell carcinoma (pancreatic endocrine), Kaposi sarcoma, Kidney (renal cell) cancer, laryngeal cancer, leukemia (eg, acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and hair cells), lip and oral cancer, liver cancer, non-small cells Lung cancer, small cell lung cancer, lymphoma (eg AIDS-related, Burkitt, skin T cells, Hodgkin, non-Hodgkin, and primary central nervous system), Waldenstrom macroglobulinemia, bone malignant fibrous histiocytoma / Osteosarcoma, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cervical cancer of unknown primary, multiple endocrine tumor syndromes, multiple myeloma / plasma cells Neoplasm, mycosis fungoides, bone marrow atypia Syndrome, Myelodysplasia / Myeloproliferative Disease, Myeloid Leukemia, Chronic Myelogenous Leukemia, Multiple Myeloma, Chronic Myeloproliferative Disease, Nasal and Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer, Mouth Pharyngeal cancer, osteosarcoma / bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, sinus and nasal cavity cancer, parathyroid cancer, Penile cancer, pheochromocytoma, pineoblastoma, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, kidney cells (kidney) Cancer, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma Squamous cell carcinoma, cutaneous T-cell lymphoma, testicular cancer, Adenoma, thymic cancer, thyroid cancer, gestational trophoblastic tumor, carcinoma of unknown primary site, cancer of unknown primary site, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma, vulva Cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

  When the antibodies of the invention or antigen-binding fragments thereof are used in combination with other therapeutically active agents, the components can be administered together or separately, sequentially or simultaneously, by any convenient route. .

  The above combinations are conveniently presented for use in the form of a pharmaceutical formulation, and therefore a pharmaceutical formulation comprising the combination described above and optionally a pharmaceutically acceptable carrier or excipient is Another embodiment of the present invention is configured. The individual components of such combinations can be administered together or separately, sequentially or simultaneously, as separate or combined pharmaceutical formulations.

  It will be appreciated that when two components are combined in the same formulation, the two components must be stable and compatible with each other and the other components of the formulation and are formulated for administration. If they are formulated separately, they can be provided as any convenient formulation, conveniently in a manner known in the art for antibodies and antigen-binding fragments thereof.

  When combined with a second therapeutic agent that is active against the same disease, the dose of each component will be different than when the antibody or antigen-binding fragment thereof is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

  Accordingly, in a further embodiment, the present invention provides a combination comprising an antibody of the invention or antigen binding fragment thereof and another therapeutically active agent.

  The combinations described above are conveniently presented for use in the form of a pharmaceutical formulation, and therefore a pharmaceutical formulation comprising the combination described above and optionally a pharmaceutically acceptable carrier is another alternative of the present invention. An embodiment is configured.

  The following examples illustrate various aspects of the present invention. These examples do not limit the scope of the invention as defined by the appended claims.

Production of Monoclonal Antibodies Monoclonal antibodies (mAbs) were produced by hybridoma cells generally according to the methods described in E Harlow and D Lane, Antibodies a Laboratory Manual, Cold Spring Harbor Laboratory, 1988. SJL mice were primed and boosted by intraperitoneal injection of RIBI adjuvant supplemented with recombinant human IGF-1R (R & D Systems, # 305-GR). The spleen of the responding animal was collected and fused with X63Ag8653GFP1L5 myeloma cells to prepare a hybridoma. Hybridoma supernatant material that binds to IGF-1R was screened using FMAT (AB18200) and BIAcore A100. AB18200 was used to bind to recombinant IGF-1R (R & D Systems, 305-GR-050 and 391-GR-050), human IGF-1R expressed in HEK293T, and cynomolgus monkey IGF-1R expressed in HEK293T. It was confirmed that it did not bind to the human insulin receptor expressed in HEK293T. BIAcore A100 was used to evaluate the binding kinetics between the antibody produced by the hybridoma and recombinant IGF-1R (R & D Systems, # 305-GR). Rabbit anti-mouse IgG (BR-1005-14, Biacore AB) was used and the antibody was captured on the chip. The hybridoma was monocloned using a semi-solid medium (methylcellulose solution), Omnitray, and the ClonePix FL apparatus.

Scale-up and purification of hybridoma material and monoclonal antibodies Scale-up hybridomas were grown in tissue culture to a scale of 4 confluent 225 cm ² flasks. At this point, cells were harvested by centrifugation at 400 g for 5 minutes. The pellet was resuspended in 100 ml serum free medium (JRH610) to wash the cells. Cells were then centrifuged at 400 g for 5 minutes. The supernatant was aspirated and discarded. The cell pellet was resuspended using 150 ml of fresh serum-free medium. Then, the cell suspension was transferred to a new 225 cm ² flask, placed in a 5-day incubator. The supernatant was then collected and centrifuged at 400 g for 20 minutes. The supernatant was collected and sterile filtered through a 0.2 μM filter in preparation for purification. The antibody was purified using a protein A resin column. The purified antibody was dialyzed against PBS at pH 7.4.

Construction of IGF-1R expression vector
Generation of an expression cassette for full length human IGF-1R The human IGF-1R cDNA expression cassette was identical to Genbank X04434 except that nucleotide 3510 was changed. As a result, the codon “GGT” of glycine 1170 is silently changed to “GGG”. Human IGF-1R cDNA was expressed from pcDNA3.1 (−) vector (Invitrogen). The sequence of human IGF-1R is set forth in SEQ ID NO: 44.

Generation of full-length mouse IGF-1R expression cassette The mouse IGF-1R cDNA expression cassette was identical to Genbank AF056187 except that nucleotide 3522 was changed. This silently changes the codon “GGC” of glycine 1174 to “GGG”. Mouse IGF-1R cDNA was expressed from pcDNA3.1D-V5-His TOPO vector (Invitrogen). The sequence of mouse IGF-1R is set forth in SEQ ID NO: 46.

Expression cassette construction of full length macaque cynomolgus monkey (Macaca fascicularis) IGF-1R The new sequence of cynomolgus monkey IGF-1R was cloned from a cynomolgus monkey kidney cDNA library by PCR. Primers were based on the human IGF-1R database entry NM_000875. PCR primers were designed with a Kozak motif at the 5 ′ end and flanking BamHI and NotI restriction sites. The BamHI-NotI PCR product was cloned into pCDNA3.1D using the vector T7 sequence proximal to the 5 ′ end of the IGF-1R coding sequence. The obtained cDNA is 99.6% identical to the human sequence at the protein level (difference 4aa from humans). The sequence of cynomolgus monkey IGF-1R is set forth in SEQ ID NO: 45.

Preparation of expression cassette for full-length human insulin receptor (type B) A DNA cassette encoding human insulin receptor type B (SEQ ID NO: 53) was cloned into pcDNA3.1 (Invitrogen). For comparison, except for the following changes, the coding sequence of SEQ ID NO: 53 is identical to the sequence shown in Genbank entry: M10051.

  Nucleotide numbering is nucleotide 1 based on the starting methionine “A” (which corresponds to position 139 of the nucleotide sequence of M10051).

Nucleotide Amino acid SEQ ID NO: 53 M10051
511 171 TAC (Tyr) CAC (His)
783 261 GAT (Asp) GAC (Asp)
909 303 CAG (Gln) CAA (Gln)
1343 448 ATC (Ile) ACC (Thr)
1474 492 CAG (Gln) AAG (Lys)
1638 546 GAC (Asp) GAT (Asp)
1650 550 GCA (Ala) GCG (Ala)
3834 1278 AAC (Asn) AAG (Lys)

  Using standard protocols and Lipofectamine reagent (Invitrogen), human, mouse, and cynomolgus monkey IGF-1R, and human insulin receptor type B vectors were transiently expressed in 293 HEK-T cells.

Recombinant protein expression and production using BacMam
Construction of pFastBacMam vector body pFastBac 1 (Invitrogen) was digested with SnaBI and Hpa1 to remove its polyhedrin promoter. This was ligated with the 3.1 kb NruI-Bst1107I fragment of pcDNA3 (Invitrogen), which fragment expresses the cytomegalovirus immediate early (CMV IE) promoter with a polylinker and a BGH polyA site, and expression of the G418 resistance gene. It contains the SV40 promoter that drives This vector allows the production of baculoviruses that express genes under the control of the CMV promoter in mammalian cells. Stable derivatives can also be selected by placing the cells under G418 selection.

A plasmid designed to express the human IGF-1R extracellular domain sequence fused to the human IGF-1R-Fc fusion protein factor Xa cleavage site and the human Fc sequence of IgG1 was constructed. The sequence encoding the extracellular domain (amino acids 1-935) of human IGF-1R cDNA was amplified by PCR and fused to the factor Xa cleavage site and the Fc sequence of human IgG1. The entire insert was then subcloned into the pFastBacMam expression vector as a HindIII-BamHI fragment. The human IGF-1R-Fc fusion protein sequence is set forth in SEQ ID NO: 47.

A plasmid designed to express the cynomolgus monkey IGF-1R extracellular domain sequence fused to the Macaca fascicularis IGF-1R-Fc fusion protein factor Xa cleavage site and the human Fc sequence of IgG1 was constructed. By cutting with Xbal and religating, the 82 bp Xbal fragment of the vector backbone was removed and the human IGF-1R expression plasmid was modified. This removes the second NotI site. The coding sequence of the extracellular domain of cynomolgus monkey IGF-1R (amino acids 1-935) was amplified by PCR as a HindIII-NotI fragment and ligated into a modified human IGF-1R expression plasmid, which removes the human sequence. Therefore, it is cut using HindIII and NotI. The sequence of the macaque cynomolgus monkey IGF-1R-Fc fusion protein is set forth in SEQ ID NO: 48.

Expression of recombinant proteins using BacMam Using the BacMam system using plasmid vectors encoding human and macaque cynomolgus monkey IGF-1R extracellular domain sequences fused to the factor Xa cleavage site and the Fc sequence of human IgG1 Protein expression was performed. Baculovirus was prepared using Bac-to-Bac system manufactured by Invitrogen. Using standard procedures, the initial PO stock was weighed into a 1 liter P1 stock. Protein production was initiated by infecting 1-5 liters of suspension culture containing HEK293-F cells with the required BacMam virus (typically performed at a multiplicity of infection (MOI) of 10 to 100: 1. However, this is usually optimized to maximize protein production). After culturing for 2-3 days, the cell culture supernatant was collected, the cells were removed by centrifugation, and then the expressed protein was purified from the clarified supernatant.

Construction of IGF-1R Ligand Expression Plasmid The gene sequences of the processed forms of IGF-I (amino acids 49-118, Swiss-prot P01343) and IGF-II (amino acids 25-91, Swiss-prot P01344) are optimal for E. coli expression Codon. The genes were freshly prepared by overlapping oligonucleotide buildup and cloned into the NdeI-BamHI site of pET-21b (Novagen). To produce a biotin-labeled IGF-1R ligand, the C-terminal 15 amino acid biotin-labeled tag sequence (GLNDIFEAQKIEWHE, see: Schatz (1993) Biotechnology (NY), 11 (10): 1138-43) SEQ ID NO: 17 Added to.

  Human IGF-I ligand and IGF-II ligand sequences are set forth in SEQ ID NO: 49 and SEQ ID NO: 51, respectively.

Expression and purification plasmid of IGF-1R ligand was transformed into E. coli BL21 (DE3) cells, then induced with 1 mM IPTG for 16 hours at 37 ° C., and then expressed using LB medium containing 100 μg / ml ampicillin. . Cell pellets were collected by centrifugation. The IGF-1R ligand was recovered by resuspending the cell pellet in 50 mM Tris (pH 8.0), 200 mM NaCl, 1 mM EDTA, 5 mM DTT, lysing by sonication, and collecting the inclusion body fraction by centrifugation. Isolated as an insoluble inclusion body. Inclusion bodies were solubilized in 50 mM Tris (pH 8.0), 6 M guanidine hydrochloride, then rapidly diluted in 100-fold excess volume of 50 mM Tris (pH 8.0), 1 mM oxidized glutathione, 1 mM reduced glutathione, followed by Soluble IGF-1R ligand was generated by mixing for 16 hours at 4 ° C. Soluble protein was concentrated and centrifuged to remove soluble material, then the biologically active IGF-1R ligand was purified by reverse phase HPLC using a Spherisorb C6 column (Waters) with an acetonitrile gradient.

Biotin labeling was performed by adding 5 mM ATP, 5 mM MgCl ₂ , 1 mM d-biotin, and 1 μM biotin ligase to the purified protein for the biotin-labeled IGF-1R ligand. The mixture was incubated at room temperature for 3 hours. Biotin-labeled IGF-1R ligand was purified by size exclusion chromatography using a Superdex 75 column (GE Healthcare). Purified IGF-1R ligand was dialyzed against PBS and quantified using a protein assay based on BSA standards and BioRad Coomassies, and then aliquots were stored at -80 ° C. The molecular weight of the purified protein was verified by mass spectroscopy. The sequences of human tagged IGF-I ligand and tagged IGF-II ligand are set forth in SEQ ID NO: 50 and SEQ ID NO: 52, respectively.

Hybridoma variable domain sequencing
Using the QIAGEN Inc. RNeasy kit (# 74106), for each hybridoma clone Total RNA was extracted from pellets of approximately 10 ⁶ cells. Promega AccessQuick RT-PCR system (A1702) was used to generate variable heavy and light chain region cDNAs using mouse leader sequences and degenerate primers specific for mouse IgG1 / _K or IgG2b / _K constant regions . TA cloning kit (Invitrogen (K2030-40)) was used to clone purified RT-PCR fragments and sequence alignment, database search, and KABAT (Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Consensus sequences were obtained for each hybridoma by alignment with known immunoglobulin variable sequences listed in Health and Human Services, National Institutes of Health (1987). Table 1 shows the numbers of the sequence listing of the variable domains of hybridomas 6E11, 9C7, 2B9, 15D9, and 5G4.

Construction of chimeric antibody A chimeric antibody containing the parental mouse variable domain grafted onto the human IgG1 / κ wild type constant region was constructed by PCR cloning. Based on the consensus sequence, primers were designed to amplify mouse variable domains, incorporating the restriction sites necessary to facilitate cloning into mammalian expression vectors. The full length heavy and light chains of the 6E11 chimeric antibody (6E11c) are shown in SEQ ID NO: 24 and SEQ ID NO: 25.

Humanized methods Humanized antibodies by the process of grafting CDR H1, CDR H2, CDR H3, CDR L1, and CDR L3 obtained from mouse 6E11 antibody and CDR L2 obtained from mouse 9C7 antibody onto appropriate human framework sequences Was made.

  The humanized variable light chain domain sequence of L0 is shown in (SEQ ID NO: 16).

  The humanized variable heavy chain domain sequences of H0 and H1 are shown in (SEQ ID NO: 14 and SEQ ID NO: 15, respectively). Optimized nucleotide sequences encoding these sequences are shown in SEQ ID NOs: 34 (H0), 35 (H1), and 36 (L0). Alternative L0 and H0 variable domain sequences are shown in SEQ ID NOs: 61 and 62, respectively, and alternative L0 light chain and H0 heavy chain sequences are shown in SEQ ID NOs: 69 and 70.

Construction PCR the humanized antibody vector using base and the method with overlapping oligonucleotides, newly constructed a DNA fragment encoding humanized variable heavy chain and variable light chain regions. PCR products were cloned into mammalian expression vectors containing human γ1 constant region and human κ constant region, respectively. This is the wild type Fc region.

  Using a similar method, the variable heavy chain region was also cloned on a variant of the human γ1 constant region containing two alanine substitutions L235A and G237A (EU index numbering). These constructs are referred to herein as IgG1m (AA). Two humanized constructs containing IgG1m (AA) variants are described as H0L0 IgG1m (AA) (SEQ ID NO: 54 and SEQ ID NO: 39) and H1L0 IgG1m (AA) (SEQ ID NO: 56 and SEQ ID NO: 39).

  Unless otherwise stated, all humanized constructs used in the examples herein include the wild type human γ1 constant region.

Expression of recombinant antibodies in CHO cells Expression plasmids encoding the heavy and light chains of chimeric or humanized antibodies, respectively, were transiently co-transfected into CHO-K1 cells. In some cases, the supernatant material was used as a test subject for binding and activity assays. In another case, the supernatant material was sterile filtered and the antibody was recovered by affinity chromatography using protein A. The antibody was also expressed in a stable polyclonal CHO cell line. DNA vectors encoding heavy and light chains were simultaneously electroporated into CHO cell suspension. Cells were subcultured in shake flasks in MR1 basal selection medium at 37 ° C., 5% CO ₂ , 130-150 rpm until cell viability and cell number improved. Subsequently, CHO cells were inoculated into MR1 basal × 2 selection medium and incubated at 34 ° C., 5% CO ₂ , 130-150 rpm for 10-14 days. Cells were pelleted by centrifugation and the supernatant was sterile filtered. Antibodies were recovered by protein A purification.

Comparative data between hybridomas and / or chimeric mAbs and / or humanized Mabs

Receptor binding ELISA
Recombinant human IGF-1R with 0.4 μg / mL histidine tag (R & D Systems, # 305-) on an ELISA plate coated with a rabbit polyclonal antibody against 6 × His (Abcam, # ab9108) at 0.5-1 μg / ml. GR-050) was captured. Anti-IGF-1R antibody from the test supernatant or purified material was titrated across the plate. Receptor binding levels were detected by treatment with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Dako, P0260) or goat anti-human kappa light chain peroxidase conjugate (Sigma, A7164). The ELISA was developed using O-phenylenediamine dihydrochloride (OPD) peroxidase substrate (Sigma, P9187).

  FIG. 1 shows binding curves for mouse antibodies 6E11, 5G4, and 15D9. FIG. 2 shows the binding curves for H0L0 and H1L0, and H0L0 IgG1m (AA) and H1L0 IgG1m (AA), confirming that they have similar binding activity when compared to the 6E11 chimera. Other monoclonal antibodies were tested (data not shown).

Receptor downregulated 3T3 / LISN c4 cells (see Mouse NIH 3T3 cell line expressing human IGF-1R, see Kaleko et al. (1990) Molecular and Cellular Biology, 10 (2): 464-473) 5 μg / mL antibody And incubated at 37 ° C. for 24 hours, then the cells were harvested. Lysates of these cell pellets were run on an SDS PAGE gel and transferred to a PVDF membrane (Western blot). Treatment with rabbit anti-IGF-1R _β C-20 antibody (Santa Cruz Biotechnology, sc-713) followed by treatment with secondary antibody (P0217) conjugated with anti-rabbit HRP, enhanced chemiluminescence (ECL) IGF-1R was detected by detection using a reagent (GE Healthcare).

FIG. 3 shows that IGF-1R _β chain is down-regulated by incubating 3T3 / LISN c4 cells with monoclonal antibody 6E11.

In a similar experiment, receptor levels were assayed in NCI-H838 cells after treatment with H0L0. NCI-H838 cells expressing human IGF-1R (1 × 10 ⁶ / well) were incubated with 5 μg / well of H0L0 for various times up to 24 hours. Cells were then harvested, cell pellets were lysed, run on a SIDS PAGE gel, transferred to a PVDF membrane, and blotted with IGF-1R using a rabbit anti-IGF-1Rβc-20 antibody (Santa Cruz, sc713). Binding was detected with an anti-rabbit HRP antibody (Dako, PO217). Western blot lanes are left-to-right, antibody-free control (taken at 24 hours), then 0, 0.2, 0.5, 1, 1.5, 3, 6, and 24 hour collections . FIG. 4 shows that H0L0 induces degradation as early as 30 minutes (0.5 hours) after exposure, but exposure lasting 3 hours is required for IGF-1R levels to reach basal levels. Show. In another experiment, 24-hour incubation of Colo205 cells with various antibodies including 6E11 parent, 6E11 chimera, H0L0 significantly reduced receptor levels as measured by Western blotting with IGF-1R β chain (Data not shown).

  In follow-up experiments, NCI-H838 lung cancer cells were treated with anti-IGF-1R antibody for therapeutic humanization. NCI-H838 cells were serum starved for 24 hours and untreated (control), or treated with 120 nM H0L0, H0L0 IgG1m (AA) or non-targeted human IgG (control) for 24 hours. Cells were washed twice with ice-cold PBS, lysed with 1% NP40 lysis buffer for 10 minutes on ice, clarified by centrifugation at 4400C for 16 minutes at 16400 rpm, and 50 μg of soluble cellular protein from 4 It was subjected to SDS PAGE reduction on a 12% polyacrylamide gel. After electrophoresis, the protein was transferred to a PVDF membrane and immunoblotted with IGF-1R or insulin receptor. To assess the loading in each lane, the membrane was also immunoblotted with α-tubulin. The amount of IGF-1R, insulin receptor, and α-tubulin were quantified using a LI COR Odyssey imaging device using a fluorescently labeled secondary antibody. The primary and secondary antibodies used were selected from Alexa680, goat anti-rabbit, molecular probe # A-21076. Use 1/20000 = 0.5 ml / 10 ml (45 ° C. for 45 minutes), IRDye800, donkey anti-mouse, Rockland # 610-732-124. Use 1/10000 = 1 ml / 10 ml (45 min at 25 ° C.) and IRDye800, donkey anti-rabbit, Rockland # 611-732-127. Use 1/10000 = 0.5 ml / 5 ml (45 ° C. for 45 minutes).

  The 97 kDa β subunit of IGF-1R, and the 200 kDa full length IGF-1R, the 95 kDa β subunit of the insulin receptor, and the 200 kDa full length insulin receptor, and the 50 kDa α-tubulin are shown in FIG. Incubation with the humanized antibody resulted in a significant decrease in IGF-1R levels. There was a reduction of about 80% compared to the sample treated with the control antibody (FIG. 5). This also resulted in a decrease in insulin receptor levels, which is likely due to degradation of the IGF-1R / insulin receptor heterodimer.

  Receptor downregulation in LISN / 3T3 c4 cells was also demonstrated using a FACS-based assay for whole blood (red blood cell depletion). H0L0 was added to whole blood samples from one donor (donor 90263) at 4 ° C. or 37 ° C. for 24 hours. FIG. 6 shows a histogram of the fluorescence intensity at 4 ° C. (solid line) and 37 ° C. (dotted line) of granulocyte and lymphocyte populations (gated using forward and side scatter profiles). Following incubation, receptor levels were assessed using PE-labeled anti-IGF-1R antibody 1H7 (BD Pharmingen, # 555999). In another experiment, HOLO or control IgG was added to whole blood samples from another donor (donor 90691) at 4 ° C or 37 ° C for 24 hours. FIG. 7 shows a histogram overlaid with the fluorescence intensity at 4 ° C. and 37 ° C. of the granulocyte population as compared to the isotype control. In both donors, incubation at 37 ° C. for 24 hours caused a significant decrease in mean fluorescence intensity compared to incubation of the same cell line at 4 ° C. Although the assay was repeated with several other donor whole blood samples, similar overall effects were obtained, but the magnitude of receptor expression and downregulation varied from donor to donor. A small number of donors had very low receptor expression and were not affected by incubation with antibodies.

Inhibition of IGF-1 or IGF-II stimulated receptor phosphorylation 96-well plates were seeded with 3T3 / LISN c4 cells at a density of 10,000 cells / well and 1-2 in complete DMEM (DMEM-Hepes modified + 10% FCS). Grown for days. Anti-hIGF-1R antibody (hybridoma supernatant or purified antibody) was added to the cells and incubated for 1 hour. 30-50 ng rhIGF-1 (R & D Systems 291-G1), or 50 ng / ml rhIGF-I (see Examples 5 and 6), or 100 ng / ml rhIGF-2 (R & D Systems 292-G2) (implementation) (See Examples 5 and 6) were added to the treated cells and incubated for an additional 20-30 minutes to stimulate receptor phosphorylation. Cells were washed once with PBS and then lysed by adding RIPA lysis buffer (150 mM NaCl, 50 mM TrisHCl, 6 mM Na deoxycholate, 1% Tween 20) + protease inhibitor cocktail (Roche 11 697 498 001). Plates were frozen for 30 minutes or overnight. After thawing, the lysate from each well was transferred to a 96-well ELISA plate pre-coated with anti-IGF-1R capture antibody (R & D Systems, MAB391) at 2 μg / ml and blocked with 4% BSA / TBS. In some experiments, another capture antibody was used (2B9 SEQ ID NOS: 10 and 11 coated at 1 μg / ml). Plates were incubated overnight at 4 ° C. Plates were washed with TBST (TBS + 0.1% Tween 20) and europium labeled anti-phosphotyrosine antibody (PerkinElmer DELFIA Eu-N1 PT66) diluted 1/2500 in 4% BSA / TBS was added to each well. After 1 hour incubation, the plates were washed and DELFIA enhancement (PerkinElmer 1244-105) solution was added. After 10 minutes incubation, receptor phosphorylation levels were measured using a plate reader setting to measure europium time-resolved fluorescence (TRF).

  FIG. 8 shows an example of receptor phosphorylation inhibition mediated by purified mouse monoclonal antibodies 6E11, 5G4, and 15D9, juxtaposing data for experiments performed simultaneously on different plates.

  FIG. 9 shows an example of inhibition of receptor phosphorylation mediated by H1L0 compared to the chimeric 6E11 antibody (6E11c).

  FIG. 10 shows an example of receptor phosphorylation inhibition mediated by H0L0 and H1L0 in connection with the wild-type IgG1 Fc region and the substituted IgG1 Fc region (IgG1m (AA)).

In another set of experiments using similar methods, IC50 values for receptor phosphorylation inhibition were obtained and humanized and 6E11 mouse parent antibodies were used for receptor phosphorylation inhibition mediated by IGF-I and IGF-II. It has been confirmed to show an equivalent profile (Table 2).

  In another set of experiments using similar methods, IC50 values for receptor phosphorylation inhibition were obtained, confirming that humanized antibody H0L0 and mouse parent antibody 6E11 show comparable activity. In parallel, 3T3 cell lines designed to express the human insulin receptor were used to test antibody activity against the insulin receptor. In this experiment, none of the antibodies inhibited receptor-induced phosphorylation induced by insulin.

Competitive ELISA
The ELISA plate was coated with anti-human IGF-1R antagonist antibody (MAB391, R & D Systems) at 2 μg / ml and blocked with 4% BSA / PBS. In the presence of purified monoclonal antibody, polyHis-tagged recombinant human IGF-1R (R & D Systems # 305-GR) was added at 400 ng / ml and incubated at room temperature for 1 hour. After the plate was washed with TBST (TBS + 0.1% Tween 20), HRP-labeled anti-poly his antibody (Sigma A7058-1 VC) was added at 12-30 μg / ml. Plates were incubated for 1 hour, then further washed and developed with OPD substrate (Sigma P9187). The reaction was stopped by adding 2M sulfuric acid and the absorbance was measured at 490 nm.

  FIG. 11A shows an example of the activity of various purified mouse monoclonal antibodies in a competitive ELISA. Data were juxtaposed for experiments performed simultaneously.

  FIG. 11B shows an example of the activity of H1L0 in a competitive ELISA compared to the 6E11 chimera (6E11c). Data were juxtaposed for experiments performed simultaneously.

  FIG. 12A shows an example of the activity of various purified humanized antibodies in a competitive ELISA compared to the mouse parent antibody (6E11) and the chimera (6E11c). In FIG. 12A, an increase in the signal of H0L0 and H0L0 IgG1m (AA) was shown compared to the repeated assays shown in FIGS. 12B and 12C.

  Figures 12B and C show examples of the activity of various purified humanized antibodies in a competitive ELISA.

Cynomolgus monkey IGF-1R binding ELISA
A 96-well ELISA plate was coated with recombinant cynomolgus monkey IGF-1R (see Example 4) overnight at 1-2 μg / ml and blocked with 4% BSA / PBS. Purified anti-hIGF-1R antibody was added and incubated for 1 hour at room temperature. The plates were washed with TBST and anti-mouse Ig conjugated with HRP (DAKO # P0260) was added to each well at 0.6-1.0 μg / ml. Plates were incubated for 1 hour at room temperature, washed with TBST, and developed with OPD substrate (Sigma P9187) or TMB substrate (Sigma T8665). The reaction was stopped with 2M sulfuric acid and the binding level was determined by measuring the absorbance at 490 nm (for OPD) and 450 nM (for TMB). For antibodies containing human IgG1 / Cκ constant regions, the anti-mouse Ig detection antibody conjugated with HRP was replaced with a goat anti-human κ light chain peroxidase conjugate (Sigma, A7164).

  FIG. 13A shows an example of the binding of a purified mouse monoclonal antibody to recombinant cynomolgus monkey IGF-1R. Data were juxtaposed for experiments performed simultaneously.

  FIG. 13B shows an example of binding of purified humanized monoclonal antibody to recombinant cynomolgus monkey IGF-1R compared to 6E11 chimera (6E11c).

Insulin receptor binding ELISA
A 96-well ELISA plate was coated with recombinant human insulin receptor (R & D Systems 1544-IR at 0.5 μg / ml overnight and blocked with 4% BSA / PBS. Purified anti-hIGF-1R antibody or mouse anti-human insulin receptor Antibody (R & D Systems MAB15441) was added to the plate, incubated for 1 hour at room temperature, then washed with TBST, anti-mouse Ig conjugated with HRP (DAKO # P0260) at 1/500 or 1 in 4% BSA / PBS The plate was added to each well and the plate was incubated for 1 hour, the plate was washed and developed by adding TMB substrate (Sigma T8665) or OPD (Sigma P9187) The reaction was stopped with 2M sulfuric acid. Binding was detected by measuring absorbance at 450 nm or 490 nm. To detect antibodies containing the normal region, the detection antibody listed above (anti-mouse Ig conjugated with HRP) was replaced with a goat anti-human kappa light chain peroxidase conjugate (Sigma, A7164).

  FIG. 14 shows an example of an insulin receptor binding ELISA using purified mouse monoclonal antibody. In contrast to the positive control antibody (R & D Systems, MAB15441), purified antibodies 6E11, 5G4, and 15D9 did not bind to the insulin receptor at concentrations up to 10 μg / ml. The experimental data were juxtaposed.

  In another experiment, similar methods were used to test the insulin receptor binding profiles of various humanized antibodies. The positive control antibody (R & D Systems, AF1544) showed good binding, but no binding between the humanized antibody and the recombinant insulin receptor could be detected at concentrations up to 50 μg / ml (FIG. 15).

Measurement of binding kinetics The binding kinetics of anti-IGF-1R antibodies to human IGF-1R was evaluated using the Biacore ^™ system. Kinetic analysis was performed using an antibody capture method. Briefly, anti-mouse IgG antibody (Biacore, catalog number BR-1005-14) was used for analysis of mouse parent antibody, and protein A was used for humanized antibody. An anti-mouse antibody or protein A was immobilized on a CM5 Biosensor chip by coupling of primary amines using the immobilized Wizard equipment attached to the instrument software according to the Biacore ^™ standard protocol (3000-4000 when immobilized normally). Resonance unit (RU) level). The anti-IGF-1R antibody was then captured directly from the hybridoma supernatant or from purified material. The capture level of the supernatant was dependent on the starting concentration of the hybridoma, which varied between about 20 RU and 650 RU. For purified material, test antibody capture levels were typically 20-600 RU. After capture, the baseline was stabilized before recombinant IGF-1R, and then R & D Systems histidine-tagged material (catalog number 305-GR) was flushed to the surface at a predetermined concentration (usually in the range of 0-256 nM). Because of the high affinity of interaction, dissociation times up to 1 hour were used. Regeneration was performed by acid elution using 100 mM phosphoric acid or 10 mM glycine (pH 1.5). Regeneration did not significantly affect the surface ability to capture antibodies in a separate analytical step. Implementation was at 25 ° C and 37 ° C. Experiments were performed on a T100 Biacore ^™ system using T100 control and analysis software. The experimental data was fit to a 1: 1 binding model associated with the instrument analysis software.

Tables 3-7 show a series of experiments performed on the supernatant and purified material.

In another experiment, the following procedure was used. Protein A was immobilized on the CM5 surface by primary amine coupling according to the manufacturer's standard protocol. Antibodies against IGF-1R were captured on this surface and allowed to stabilize for a period of time before recombinant human or cynomolgus monkey IGF-1R was injected onto the captured surface. In general, the concentration of IGF-1R used was 256-16 nM, but 0 nM injection (buffer only) was also used for the double reference to best perform Biacore kinetic analysis. Data were analyzed using the 1: 1 model associated with the Biacore instrument and the work was performed at 37 ° C. using HBS-EP running buffer at T100. Humanized mutants H0L0 and H0L0 IgG1m (AA) show high affinity binding (about 300-600 pM) to recombinant human and cynomolgus monkey IGF-1R, confirming results indicating kinetics comparable to 6E11 chimera Has been.

  Similar experiments were performed to confirm that the H0L0, H0L0 IgG1m (AA) and 6E11 chimeras showed comparable binding kinetics. However, the overall affinity was lower than that of all previous experiments (1.88, 1.84, and 1.52 nM, respectively). The reason for the obvious difference is unknown.

Inhibition of ligand binding quantified using Biacore Experiments were performed using two different densities of capture biotin-labeled IGF-I. Briefly, 200 or 4000 RU was stably captured on a streptavidin sensor chip. To test the neutralizing ability of anti-IGF-1R antibodies, different concentrations of antibody were premixed with a fixed concentration of recombinant IGF-1R. As a control, unbiotinylated IGF-1 was also mixed with the same concentration of IGF-1R. The mixture was then run over the IGF-I surface and the maximum association point was measured. This reading was then compared to a sample containing the same concentration of his-tagged IGF-1R in the absence of anti-IGF-1R antibody. The presence of neutralizing antibodies blocked the binding of IGF-1R and IGF-I and reduced the maximum association observed. The inhibition rate (%) was calculated by comparing the values. Regeneration was carried out using 2 additions of 4M magnesium chloride. Experiments were performed on a Biacore 3000 apparatus.

Tables 9 and 10 detail the inhibition rates obtained and further the concentrations of antibodies IGF-1 and IGF-1R used to obtain these results.

In another experiment, captured biotin-labeled ligands (IGF-I, IGF-2) were used and the ability of humanized antibodies to directly block ligand binding was assessed using a Biacore-based method. Biotin-labeled IGF-1 or IGF-2 was immobilized at about 300 RU and 350 RU, respectively, on a streptavidin biosensor chip. IGF-1R was flushed to the surface with 50 nM alone or with 50 nM premix containing 250 nM anti-IGF-1R antibody (for IGF-I experiments) or 500 nM anti-IGF-1R antibody (for IGF-2 experiments) . IGF-1R binding was also performed in the presence of the natural ligands IGF-1 and IGF-2 (both biotin unlabeled). The surface was regenerated using 100 mM phosphoric acid. Experiments were performed at 25 ° C. with a Biacore 3000 using HBS-EP buffer. Note: Analysis of IGF-2 assay data was complicated by the fact that some of the antibodies showed non-specific binding with the IGF-2 surface alone. For immobolised IGF-I, the most efficient receptor binding inhibitor was unlabeled IGF-I, with IGF-2 and H0L0 showing approximately 60% inhibition (Table 11). For immobilized IGF-2, the most efficient receptor binding inhibitor was unlabeled IGF-I or IGF-2. This assay showed partial inhibition of IGF-2 binding by neutralizing antibodies including H0L0 (Table 11).

Fluorescence activated cell sorting (FACS) analysis Colo205 cells (2 × 10 ⁷ cells / ml) were stained with 10 μg / ml anti-hIGF-1R purified antibody in FACs buffer (4% FCS / PBS) for 1 hour. . Cells were also stained with a suitable negative control mouse antibody (Sigma # 15154). Cells were washed with FACS buffer and then stained with anti-mouse IgG PE secondary antibody 1: 100 (Sigma P8547). After washing with FACS buffer and fixing with Cell Fix (Becton Dickinson), the cells were analyzed by flow cytometry.

  FIG. 16 demonstrates that antibody 6E11 can recognize IGF-1R naturally expressed on the surface of human tumor cell lines.

  In another experiment, humanized antibodies were tested for their ability to stain various human tumor cell lines known to overexpress IGF-1R. NCI-H838 lung cancer cells were stained with 100 μg / ml of the selected antibody for 45 minutes at 4 ° C. Binding was detected with an anti-human IgG antibody conjugated with PE (Sigma P8047). Samples were analyzed by flow cytometry using a Becton Dickinson FACscan cytometer. The results shown in FIG. 17 confirm that the humanized variant can bind to NCI-H838. Similar results have been obtained with MCF7 breast cancer cells and A549 lung cancer cells (FIG. 18).

Immunohistochemistry with frozen tissue sections Tissue sections were placed on glass slides, fixed with acetone for 2 minutes, and then loaded into an automatic slide stainer (Dako Cytomation S3400). Slides were then blocked and stained with mouse antibody (primary antibody) and anti-mouse Ig-HRP secondary antibody (DakoCytomation Envision Kit) using standard immunochemical staining methods. After this secondary incubation, the slides were washed and developed using DakoCytomation Envision DAB solution, rinsed, dehydrated, and covered with a coverslip for viewing. An unrelated control antibody (mouse IgG1 purified from MOPC21 hybridoma) was used as a negative control.

  Similarly, humanized and chimeric antibodies were analyzed except that these antibodies were biotinylated to facilitate detection. However, the presence of the biotin tag was found to reduce the activity of these antibodies as measured by ELISA (data not shown), thus increasing the concentration of primary antibody used to 100 μg / ml. The secondary antibody (DakoCytomation Envision Kit-anti-mouse Ig-HRP conjugate) listed above was replaced with streptavidin-HRP (DakoCytomation catalog number 1016). Another unrelated antibody was also biotin labeled and used as a negative control (Sigma # 15154).

Samples were analyzed as follows. After calibrating the instrument using a calibration carrier (# 69935000, 05041103097), the slides were loaded into a ChromaVison automated cell imaging device and scanned 10x. Data analysis was performed to calculate the tissue staining rate (%) (defined as brown / brown + blue ^* 100).

  Figures 19 and 20 show that 6E11 stains human tumor tissue samples. A positive control antibody was included as a reference (Abcam, # 4065).

  FIG. 21 shows that 6E11 chimera (6E11c) and H1L0 stain human tumor tissue samples.

  In a separate experiment, a frozen tissue microarray (Array ID: MB-1002) obtained from Cytomyxx was used to test the ability of humanized H0L0 to recognize human IGF-1R on human tumor tissue samples. The array includes 10 lung tumor cores, 10 breast tumor cores, 10 colon tumor cores, and 10 prostate tumor cores. H0L0 was labeled with biotin to facilitate detection. Frozen microarray slides (Cytomyxx MB-1002) were fixed with acetone / ethanol (50:50) solution at 4 ° C. for 5 minutes, washed and then treated with 3% hydrogen peroxide for 5 minutes to remove any endogenous peroxidase. Was also removed. Slides were stained with 7.0 μg / ml biotin-labeled anti-IGF-1R H0L0 for 1 hour at room temperature. After washing, streptavidin peroxidase was applied for 20 minutes and visualized with DAB (diaminobenzidine) for 2 minutes. Finally, the sections were washed and counterstained with Mayer hematoxylin, rinsed with tap water, dehydrated and clarified. The tumor samples are as follows: lung (right: squamous cell tumor, left: adenocarcinoma); breast (right: adenocarcinoma, ductal epithelium, left: adenocarcinoma, invasive); colon (right) : Well-differentiated adenocarcinoma, left figure: well-differentiated adenocarcinoma); prostate (right figure: adenocarcinoma, left figure: adenocarcinoma).

The results confirm that H0L0 stained the viable sections moderately / highly as summarized in Table 12. Representative high magnification images (200 ×) of lung, breast, colon, and prostate tumors are shown in FIG. 22, confirming that the biotin-labeled H0L0 antibody stains epithelial cells extensively.

Inhibition of AKT signaling
Costar 96-well plate (# 3598) was coated with 50 μl of PBS solution containing 2% gelatin and incubated in a 37 ° C. incubator for at least 1 hour. Prior to use, the plate was rinsed once with PBS. Primary human preadipocytes were trypsinized, centrifuged, and the medium was siphoned off. Cells were resuspended in 10 ml of warm preadipocyte growth medium (ZenBio, # PM-1). The cell density was adjusted to 150,000 cells per ml of preadipocyte growth medium (ZenBio). Two T225 Costar flasks containing 50 ml of medium were seeded with 1 million cells. Utilizing a Multidrop 384 or similar instrument, the remaining cells were used to seed gelatin coated 96 well plates (100 μl = 15,000 cells / well). Cells were incubated overnight at 37 ° C., 5% CO ₂ atmosphere, 90% humidity. The next day, the medium was removed, 200 μl of induction medium was added, and the plate was covered with Breath-Easy gas permeable film (Sigma # Z380059). Plates were incubated for 6 days at 37 ° C., 5% CO ₂ atmosphere, 90% humidity. After 6 days, the medium was aspirated and 200 μl of differentiation medium was added. The plate was covered with Breath-Easy gas permeable film and incubated for 7 days at 37 ° C., 5% CO ₂ atmosphere, 90% humidity. After cell differentiation, the medium was aspirated and the cells were seeded once with 200 μL PBS. 75 μl of adipocyte starvation medium was added, the plates were coated and incubated overnight at 37 ° C., 5% CO ₂ atmosphere, 90% humidity. The test samples were diluted with 4 times the final concentration of adipocyte starvation medium. 25 μL of diluted test compound was added to each well and incubated at 37 ° C. for 1 hour. IGF-I ligand (R & D Systems, # 291-G1) was diluted to 30 nM with adipocyte starvation medium and 20 μL of 30 nM IGF-I was added to each well (final concentration 5 nM). After a period of time when the supernatant was removed by flicking the medium into the sink, the plates were incubated at 37 ° C. for exactly 5 minutes. The plate was dried on a paper towel.

  65 μl of complete lysis buffer (MSD lysis buffer containing phosphatase and protease inhibitors) was added to each well and the plate was sealed with a heated plate sealer. MSD assays were performed after storing the plates at −80 ° C. (for later analysis) or placing them on a shaker (approximately 500 rpm) for 15 minutes at room temperature.

  The level of phosphorylated AKT (pSer473) was assessed using the MSD phosphorylation assay kit (# K111 CAD). Briefly, 150 μL / well blocking solution (MSD blocker A dissolved in MSD Tris wash buffer) was added to each well of the MSD assay plate. The plate was sealed and placed on a shaker at 300 rpm for 1 hour at room temperature using a bench top plate shaker. Blocking solution was removed from the MSD plate and the plate was washed 4 times with 200 μL / well of 1 × MSD Tris wash buffer. 50 μL / well of cell lysate from the cell plate was transferred to the corresponding well of the MSD plate and sealed. Using a bench top plate shaker, the plates were shaken at 300 rpm for 1 hour at room temperature. The MSD plate was washed 4 times with 200 μL / well using 1 × MSD Tris wash buffer (ELx405).

  25 μL of the diluted detection antibody mixture (final concentration 10 nM) was added to each well of the MSD plate. Using a bench top plate shaker, the plates were shaken at 300 rpm for 1 hour at room temperature, then washed 4 times with 200 μL / well using 1 × MSD Tris wash buffer (ELx405). 150 μL of Read Buffer T containing surfactant was added to each well, and the plate was read with an MSD 6000 SECTOR reader. The signal intensity decreased with reading buffer over time, but the signal window typically remained stable for about 20-30 minutes.

Table 13 shows a summary of data from three independent plates, showing that purified mouse parent, chimera, and humanized Mab inhibit IGF-I mediated induction of AKT phosphorylation. Plates 1 and 2 were run in parallel. Another day, plate 3 was performed. The value is expressed as pIC50 (= −log10 (IC50) (g / ml)).

In two separate experiments, various humanized antibodies were tested for inhibition of AKT phosphorylation in response to IGF-I or insulin stimulation. For the IGF-I results shown in Table 14, values are expressed as mean pIC50 [g / ml; pIC50 = −log10 (IC50)]. Vmax = maximum inhibition expressed as a percentage of signal in the absence of ligand. Experiment 1 is an average of 4 runs. Experiment 2 is the average of 3 runs. Experiments 1 and 2 were conducted with an interval of about 6 months. Several antibodies (** H0L0) were tested with different batches of material in parallel. Compare with negative control antibody. All anti-IGF-1R antibodies showed a dose-dependent inhibition of AKT phosphorylation by humanized antibody H0L0, showing activity comparable to 6E11 mouse parent antibody or 6E11 chimeric antibody (Table 14).

  Since this assay system was also sensitive to insulin-mediated phosphorylation of AKT (via the insulin receptor), the effect of the humanized antibody on insulin signaling was paralleled with the IGF-I experiment described above. evaluated. In contrast to the results from IGF-I stimulation, the humanized antibody showed no inhibition of insulin receptor signaling beyond the non-specific effects observed with the negative control antibody (data not shown). . These results were observed in 2 independent experiments (total of 7 runs).

Proliferation assay using MCF7 cells MCF-7 cells (ATCC HBT-22) were seeded in 96-well plates at a density of 10,000 cells / well, and complete medium (MEM + Earles salt + 10% FCS + 0.1 mg / ml bovine insulin (Sigma 10516)). ) For 2 days. Cells were washed and incubated with serum free MEM (serum free, insulin free) for 4 hours. The medium was removed and replaced with a range of concentrations of purified antibody (0.014-10 μg / ml) diluted in serum-free medium (100 μl / well). After incubating the cells for 1 hour, IGF-I (R & D Systems # 291-G1) was further added to a final concentration of 50 ng / ml. All treatments were performed in triplicate. Cells were incubated for 5 days at 37 ° C., 5% CO ₂ . After incubation, 15 μl of MTT dye solution (Promega # G402A) was added to each well and the plate was incubated for an additional 4 hours. 100 μl of stop / solubilization solution (Promega # G401A) was added to each well and the plate was gently shaken overnight at room temperature. The next day, proliferation levels were measured by measuring absorbance at 570 nm using a plate reader.

  FIG. 23 shows the activity of various purified mouse monoclonal antibodies that inhibit tumor cell growth. Data were collocated for the experiment.

Proliferation assay- LISN cells LISN cells (3T3 hIGF-1R) were seeded at a density of 10,000 cells / well in white wall 96-well plates (Corning 3610) and grown in complete medium (modified DMEM-Hepes + 10% FCS) for 1 day. It was. The medium was removed and the cells were incubated with serum free DMEM for 4 hours. The medium was removed and replaced with a range of purified antibodies (0.0041-3 μg / ml final concentration) diluted in serum-free medium (50 μl / well). After incubating the cells for 1 hour, an additional 50 μl / well of IGF-1 (R & D Systems 291-Gl or IGF-I; see Examples 5 and 6) was added to a final concentration of 50-60 ng / ml. All treatments were performed in triplicate. Cells were incubated for 0-3 days at 37 ° C., 5% CO ₂ . After incubation, 100 μl of freshly prepared Promega CellTitre-Glo reagent (Promega G7571) was added to each well and the plate was shaken for 2 minutes. The plate was further incubated at room temperature for 10 minutes to stabilize the signal, and then the luminescence signal was measured with a Wallac Victor plate reader.

  Figures 24 and 25A-E show the activity of purified 6E11 mouse monoclonal antibodies, 6E11c H0L0 and H0L0 IgG1m (AA), and H1L0 and H1L0 IgG1m (AA). The data confirms that H0L0 and H1L0 can inhibit tumor cell growth in vitro. Data were collocated for the experiment.

Cell Cycle Inhibition NCI-H838 (ATCC CRL-5844) cells were seeded in 24-well microplates at a density of 2 × 10 ⁵ cells / well and grown overnight in 1 ml complete RPMI (RPMI + 10% FCS). The next day, the cells were washed with SFM (serum free RPMI medium) and incubated in 1 ml of the same medium for 4 hours. The medium was aspirated from the cells and 500 μl of SFM containing 20 μg / ml purified antibody was added (10 μg / ml final concentration). Cells were incubated for 1 hour. To several wells, SFM containing IGF-I (R & D Systems 291-G1) was added to a final concentration of 50 ng / ml. Treated cells were incubated overnight. The next day, cells were gently washed with PBS and then harvested by adding 200 μl of Versen solution (Invitrogen # 15040). The cell suspension was transferred to a 96 well V-bottom plate. After pelleting the cells by centrifugation, the cells were fixed by adding chilled 80% ethanol and incubated on ice for 30 minutes. Cells were pelleted and resuspended in 200 μl of 50 μg / ml propidium iodide, 0.1 mM EDTA, 0.1% Triton X-100, 0.05 mg / ml RNAse A. Cells were incubated on dark ice until analyzed by flow cytometry.

  FIG. 26 shows the cell cycle state of various treatment groups in the presence of IGF-I, in which the cells are induced to cycle. In the presence of 6E11 antibody, the cell cycle was inhibited at a level comparable to that of cells incubated in the absence of IGF-I.

Protection from apoptosis 96-well microplates were seeded with NCI-H838 cells (ATCC CRL-5844) at a density of 10,000 cells / well in 100 μl complete RPMI medium and allowed to grow for 2 days. The cells were then washed with SFM (RPMI-free serum) and incubated with 100 μl SFM for 4-5 hours. After removing the medium, it was treated with no antibody, negative control antibody, or 20 μg / ml purified anti-hIGF-1R antibody (6E11). Cells were further treated with SFM alone, SFM + 20 ng / ml IGF-1, SFM + 5 μM camptothecin, or 5 μM SFM + camptothecin + 20 ng / ml IGF-1. All treatments were tested in triplicate with a final volume of 100 μl. The plate was then incubated for 20 hours. The medium was aspirated from the wells and the cells were lysed by adding 200 μl of PBS containing 0.5% NP-40, followed by incubation for 5 minutes with shaking at room temperature. 20 μl of lysate was transferred to a prepared microplate of the Roche cell death ELISA kit and 80 μl of incubation buffer was added. Absorbance at 405 nm was measured using a microplate reader according to the protocol described in the kit insert (Roche catalog number 1 544 675).

  FIG. 27 shows that the presence of IGF-I protects NCI-H838 cells to some extent from camptothecin-induced apoptosis. Addition of 6E11 reversed IGF-1-mediated protection from apoptosis.

In another experiment, selected antibodies were tested for their ability to prevent IGF-1 rescue from camptothecin-induced apoptosis in A549 cells. A549 cells were seeded at 1 × 10 ⁴ , grown in 96-well plates, and treated with 20 μg / ml of the selected antibody for 1 hour in serum-free conditions. 15 ng / ml IGF-1 and 5 μg / ml camptothecin were added together and the cells were incubated overnight. The level of apoptosis was measured using a Roche cell death detection ELISA kit (Roche 11774425001) that evaluates the relative level of DNA fragmentation. As shown in FIG. 28, all humanized antibodies prevented rescue from IGF-1 induced apoptosis.

Absence of agonist activity in the presence or absence of cross-linked antibody 96-well microplates with 3T3 / LISN c4 cells at 10,000 cells / well in complete DMEM (DMEM Hepes modification + 10% FCS) Seeded at density and grown for 2 days. Purified anti-IGF-1R antibody was titrated into cells in complete DMEM, but each dilution was tested in triplicate. Antibodies reported to have agonist activity (# 556000, BD Biosciences) and / or 50 ng / ml IGF-I (20-30 min incubation) were included in some experiments as positive controls. Non-related antibodies and media alone negative controls were included. In another experiment, anti-mouse cross-linked antibody (Sigma M8144) or anti-human cross-linked antibody (Sigma 13382) was included with antibody titration at a ratio of 2: 1 [anti-IGF-IAb]: [cross-linked Ab]. The plate was incubated for 30 minutes. After aspirating the medium and gently washing the cells once with PBS, RIPA lysis buffer (150 mM NaCl, 50 mM TrisHCl, 6 mM Na deoxycholate, 1% Tween 20) + protease inhibitor cocktail (Roche 11 697 498 001) Used to dissolve. The plate was left at -20 ° C overnight. After thawing, 100 μl of the lysate sample was transferred to a 96-well ELISA plate pre-coated with 2 μg / ml anti-IGF-1R capture antibody (2B9) and blocked with 4% BSA / TBS. Plates were incubated overnight at 4 ° C. Plates were washed 4 times with TBST (TBS + 0.1% Tween 20) and europium labeled anti-phosphotyrosine antibody (DELFIA Eu-N1 PT66, PerkinElmer) diluted 1/2500 in 4% BSA / TBS was added to each well. After 1 hour incubation, the plates were washed as before and 100 μl DELFIA enhancement solution (PerkinElmer 1244-105) was added. After 10 minutes incubation, receptor phosphorylation levels were measured using a plate reader set up to measure europium time-resolved fluorescence (TRF).

  FIG. 29 shows that 6E11 has no agonist activity at concentrations up to 10 μg / ml in the presence of cross-linked antibody. Data were collocated for the experiment.

  In another experiment, a preadipocyte assay system was used to determine whether the 6E11 series of humanized anti-IGF-1R antibodies modulates phospho-AKT basal levels that are believed to exhibit agonist properties. In this experiment, preadipocytes were differentiated and processed as described in Example 21. However, the stimulation step was omitted to assess the basal level of AKT phosphorylation in the presence of humanized antibody. The results show that basal AKT phosphorylation did not increase with humanized antibodies at concentrations up to 20 μg / ml (FIG. 30).

  In a parallel experimental set using lung cancer cell line A549, basal AKT phosphorylation levels were assessed in the presence of 0-20 μg / ml antibody and in the absence of ligand. Two different H0L0 batches, a negative control sample, and 11C11 (antibodies showing moderate activation of receptor phosphorylation, see FIG. 31). The y-axis shows the AKT phosphorylation level in arbitrary units. Consistent with the data presented in Example 21, H0L0 inhibited IGF-I-mediated Akt phosphorylation in a dose-dependent manner (IC50 ranges from 200 to 500 ng / ml, data not shown). However, increasing the antibody concentration in the absence of ligand appears to slightly increase the basal level of phospho-Akt. However, the signal is a plateau and appears to reach only about three times the resting level with two different material batches. The reason why the signal is somewhat increased is unknown and this data is not supported by any other experimental data.

  In a parallel experimental set using LISN-c4 3T3 cells, the effect of humanized antibodies on basal levels of receptor phosphorylation was evaluated in the absence of ligand stimulation. LISN cells were incubated for 30 minutes in the presence of a range of concentrations (27 ng / ml to 20 μg / ml) of the selected antibody. Titrations of known agonist antibodies 11C11 and BD556000 (Becton Dickinson) were also included. Control wells stimulated with 100 ng / ml IGF-2 can be included. IGF-1R phosphorylation was measured using the DELFIA assay described above. In contrast to the two positive control antibodies 11C11 and BD556000 that induced a dose-dependent increase in receptor phosphorylation, there was no increase in basal levels of phosphorylated receptors with humanized antibodies (FIG. 32). Since cross-linking of surface-bound antibodies can induce receptor signaling, the experiment was repeated in the presence of cross-linked antibodies. Selected antibodies mixed with suitable cross-linked antibodies in a 2: 1 ratio were incubated with LISN cells at a range of concentrations for 30 minutes (Sigma 13382 for humanized antibodies and Sigma M8144 for mouse antibodies). . IGF-1R phosphorylation was measured using the DELFIA assay described above. The results show that the humanized antibody did not raise the basal level of phosphorylated receptor, in contrast to 11C11 which raised the phosphorylated receptor level (FIG. 33).

The effect of addition of humanized antibody in the absence of ligand on the growth of LISN-c4 and NCI-H929 (human multiple myeloma cell line) was also tested. NCI-H929 cells were seeded at 4 × 10 ⁴ cells / well in serum-free medium in 96-well plates. Dilutions of selected antibodies were added in the range of 20-0.019 μg / ml. After incubating the cells for 4 days at 37 ° C., proliferation was measured using the Promega Cell Titre Blue assay kit (Promega G8081). FIG. 34 shows that H0L0 does not stimulate the growth of NCI-H929 cells in serum-free medium. Similar results were observed with LISN-c4 cells (data not shown).

Allograft model-3T3 / LISN c4
An in vivo tumor model using 3T3 / LISN c4 cells was used to establish the ability of 6E11 mouse monoclonal antibodies to inhibit the growth of pre-established tumors in athymic nude mice. Tumors were induced by methods similar to those published in Cohen et al., Clinical Cancer Research 11: 2063-2073 (2005). Briefly, 4-6 weeks old athymic CD1 nu / nu mice were inoculated subcutaneously with 2.5 × 10 ⁶ LISN cells suspended in 0.1 ml Matrigel ^™ . When the tumor size reached approximately 150 mm ³ , mice were treated by intraperitoneal injection of 0.2 ml PBS containing 250 μg of antibody twice a week for 3 weeks. Tumors were measured at two diameters with calipers three times a week and the volume was calculated using the formula (length × [width] ² ) / 2. Data was analyzed as follows: Volume of Log ₁₀ transformed tumors was analyzed using random coefficient regression analysis. This estimates the intercept (baseline) and slope (tumor growth rate) for each group. Compared to the PBS treated group, the 6E11 group had a 31% decrease in growth rate (FIG. 35, p = 0.007).

In a similar experiment, nude mice were implanted subcutaneously with Matrigel containing 2.5 × 10 ⁶ cells. On day 18 after transplantation, mice with tumor volumes of 100-200 mm ³ were randomly divided into groups of 8 animals per treatment group. Anti-IGF-1R antibody 6E11 was administered by intraperitoneal injection at a dose of 250 μg / mouse and 100 μg / mouse twice weekly for 3 weeks. Control animals received saline on the same schedule. Tumor size and mouse body weight were measured twice a week. A decrease of 56% and 70% in tumor volume measured on day 35 compared to the saline treatment group was seen in the 100 μg / mouse and 250 μg / mouse groups, respectively (FIG. 36).

In another study, various antibody activities were tested compared to a PBS negative control group. Each CD1 nu / nu athymic mouse is implanted subcutaneously with 2.5 × 10 ⁶ LISN / 3T3-c4 cells suspended in Matrigel, the tumor size is measured with calipers, and the volume is represented by the equation: tumor volume = length Calculated by length × (width) ² × 0.5. When tumors reached an average volume of about 150 mm ³ , animals were randomly divided into groups with equivalent tumor sizes, and each animal was injected intraperitoneally with PBS containing 250 μg of a suitable monoclonal antibody twice a week for 3 weeks. Gave. The treatment groups were two different batches: 6E11 (mouse parental mAb), H0L0 IgG1m (AA) and H0L0. PBS-only treatment was used as a vehicle control for these studies. The group size for this study was a minimum of 14. Data were expressed as mean tumor volume ± standard error relative to days after antibody treatment. Both 6E11 and H0L0 showed a statistically significant decrease in tumor growth rates of 52% (P <0.0001) and 60% (P <0.0001), respectively (FIG. 37). Consistent with previous studies (data not shown), neither batch of H0L0 IgG1m (AA) antibody significantly altered tumor growth rate compared to the PBS control group.

  In addition to the reduction in tumor growth rate, time-to-cull survival was improved in mice treated with 6E11 and H0L0 compared to PBS control (data not shown). Consistent with the tumor growth data, H0L0 IgGm (AA) did not benefit from delayed exclusion time (data not shown).

  This study was repeated using 6E11 and H0L0 antibodies, but no matrigel was used. The results of this study reflect the results of previous studies, compared to the PBS control, 6E11 and H0L0 have tumor growth rates of 20% (p = 0.0464) and 29.7% (p = 0), respectively. .0037), indicating a significant decrease. The exclusion time survival of the group of mice treated with 6E11 and H0L0 was also improved compared to the PBS control. In this experiment, an unrelated antibody was used as a control and showed a profile similar to the PBS group (data not shown).

Inhibition of Colo205 Cell Tumor Growth by 6E11 Mouse Parent Antibody An in vivo tumor model using Colo205 cells was used to establish the ability of 6E11 mouse monoclonal antibodies to inhibit the growth of tumors previously established in HRLN female nu / nu mice. 1 × 10 ⁶ Colo205 cells were suspended in 50% matrigel and transplanted subcutaneously into the flank of nude mice. When the tumor size reached approximately 80-120 mm ³ (equivalent to day 1 in FIG. 38), the mice were treated with 10 mg / kg antibody by intraperitoneal injection every 3 days for a total of 10 injections. Tumors were measured with calipers and volume was calculated using the formula (length × [width] ² ) / 2. Data were analyzed as follows: Log ₁₀ transformed tumor volumes were analyzed using random coefficient regression analysis. This evaluates the intercept (baseline) and slope (tumor growth rate) of each group. Compared to vehicle control (PBS), 6E11 antibody reduced the growth rate by 58% (Figure 38, p = 0.0019).

  An experiment similar to that described above was performed on another occasion. However, in this second experiment using Colo205 cells, inhibition of tumor growth was not observed in animals treated with 6E11. The reason why no inhibition was observed with 6E11 is unknown (data not shown).

In addition, an experiment similar to the above experiment was performed using mice transplanted with 1 × 10 ⁷ A549 cells. However, in this experiment, inhibition of tumor growth was not observed in animals treated with 6E11. The reason why no inhibition was observed with 6E11 is unknown (data not shown).

  From these last two experimental data, it appears that the antibodies of the invention do not inhibit tumor growth in these models, but the first two tumor models (the allograft model and the first colo205 model) are Considered more robust. Control antibodies that gave a positive signal in these first two models (ie, showed inhibition of tumor growth) showed no inhibition in the second Colo205 tumor model study or the A549 tumor model study. The inventors are therefore more confident that the data from the first two tumor models show more test antibody activity than the data from the second two models.

  Three additional xenograft studies were performed with 6E11 or a humanized variant thereof. In the first study, the activity of 6E11 was compared to H0L0 IgG1m (AA) in the Colo205 model. In contrast to the results shown in FIG. 38, there was no indication of inhibition of tumor growth after treatment with 6E11 or H0L0 IgG1m (AA) compared to the PBS control group. In repeated studies where the treatment groups were PBS, 6E11, H0L0, positive control antibody, and unrelated antibody control, none of the groups showed any signs of inhibiting tumor growth, but tumor growth compared to PBS Exclude positive controls that showed a 23% reduction in rate (p = 0.003). However, the unrelated antibody control showed 15% tumor growth inhibition compared to PBS (p = 0.053). There was no inhibition of tumor growth with any antibody compared to the unrelated antibody control.

  In another xenograft model (MCF-7 breast tumor model), animals were treated with PBS, 6E11, H0L0, positive control antibody, and an unrelated antibody control. In this study, no treatment group was separated from PBS or unrelated antibody controls. In both studies, treatment with paclitaxel significantly inhibited tumor growth (data not shown).

  For post-study analysis, tumor samples taken at the end of all three studies were evaluated for receptor expression by immunohistochemistry. No signs of IGF-1R receptor expression were observed using the biotin-labeled H0L0 probe, but the same labeled antibody stained the LISN / 3T3 c4 tumor sample and the patient tumor sample positively.

  The first Colo205 study shown in FIG. 38, the tissue results obtained from the LISN model (IGF-1R-11 and IGF-1R-12 studies), and the three species that did not show antibody-mediated tumor growth inhibition The reason for the apparent difference in antibody activity between results obtained from additional studies is unclear. However, the in vivo efficacy of anti-IGF-1R antibodies is assessed from the fact that tumors derived from at least some of the Colo205 and MCF-7 cell lines are receptor negative (including the PBS control group) at the end of the study. There are concerns about the validity of these particular experiments.

In order to examine the reappearance of the receptor after the withdrawal of the antibody, NCI-H838 cells were grown in growth medium in the presence of 1.67 μg / ml H0L0 (H0L0) antibody shown in FIG. Incubated with. A first cell sample was taken 0.5 hours after antibody addition. Three hours after antibody addition, all other samples were washed thoroughly with PBS before returning to growth medium. Cells were then harvested 3, 4, 5, 6, 7 and 24 hours after antibody addition and evaluated for IGF-1R expression by Western blot using rabbit anti-IGF-1Rβ c20 antibody (Santa Cruz, sc713). did. Binding was detected using HRP antibody and IgG1 kappa was used as a negative control antibody. Lanes 1-7 are harvested at 0.5, 3, 4, 5, 6, 7, 24 hours. Lanes 8 and 9 are antibody-free control and negative control antibody (Sigma 15154), respectively, and were collected at 3 hours. The Magic Mark (Sigma, LC5602) is shown in lane 10. After antibody removal (t = 3 hours), Western blot shows no signs of IGF-1R expression at t = 7 hours (lane 6). On the other hand, t = 24 h (lanes 7, 21 hours after antibody removal) to have appeared strong band matching the IGF-1R _beta chain.

  A second experiment (data not shown) confirmed that receptor reappearance was maintained at 48, 72 and 96 hours. These data suggest that the reappearance of most receptors occurs within the first 4-21 hours after antibody removal.

IGF-1R / IR heterodimer binding assay In cells expressing both IGF-1R and insulin receptor (s), it is shown that the hybrid receptor IGF-1R: insulin receptor (InsR) is present (Pandini et al., (1999) Clin Cancer Res., 5 (7): 1935-44). Recently, anti-IGF-1R antibodies that do not cross-react with insulin receptors have been shown to reduce insulin receptor levels, possibly by internalizing and degrading hybrid receptors (Sachdev (2006) Cancer Res., 66 (4): 2391-402).

  Humanized antibodies were evaluated for activity against hybrid receptors using a co-immunoprecipitation assay. In the first experiment shown in FIG. 40A, COLO-205 colon cancer cells and recombinant NIH-3T3 cells expressing the human insulin receptor were lysed with 1% NP40 lysis buffer for 10 minutes on ice and 16400 rpm at 4 ° C. And clarified by centrifugation for 20 minutes. Immunize soluble cellular protein (500 μg) with 5 μg of antibody against IGF-1R (6E11, 6E11 chimera, H0L0 IgG1m (AA), IGF-1Rβ, insulin receptor (β), or non-targeted human IgG (control) Immunoprecipitated proteins were subjected to reducing SDS PAGE on 4-12% polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with IGF-1R or insulin receptor, using fluorescently labeled secondary antibodies Then, an IGF-1R / insulin receptor immunoblot was imaged using the LI COR Odyssey system, in another experiment using the similar method described above (FIG. 40B), comparing H0L0 and H0L0 IgG1m (AA). The unbound fraction is added to the material in the supernatant after immunoprecipitation. It is noted that the 97 kDa β subunit of IGF-1R and the full length IGF-1R of 200 kDa, and the 95 kDa β subunit of the insulin receptor and the full length insulin receptor of 200 kDa are shown in the figure. As shown in Figures 40A and 40B, the humanized antibody was able to co-immunoprecipitate insulin receptor and IGF-IR from the Colo205 human carcinoma cell line at a level comparable to the 6E11 chimera. In the absence of IGF-1R, the insulin receptor was not immunoprecipitated (see FIG. 40A, fourth figure, lanes 1-5), indicating that the antibody is not reactive to the insulin receptor. After immunoprecipitation with an anti-IGF-1R antibody, most of the IGF-1R is removed from the cell lysate and human It was further shown that the conjugated antibody can bind to heterodimeric IGF-1R: insulin receptor and homodimeric IGF-1R receptor (FIG. 40B, top lanes 3-5), but cells after IP In the lysate, a significant proportion of insulin receptors (which would be mostly homodimeric moieties) remained (FIG. 40B, lower lanes 3-5).

Growth of NCI-H929 cells Human myeloma cell line NCI-H929 was stimulated with IGF-1 in the presence of various concentrations of selected antibodies in serum-free medium. NCI-H929 cells were washed with serum-free medium and seeded at 4 × 10 ⁴ cells / well in a 96-well plate. A dilution of the selected antibody was added in the range of 20-0.019 μg / ml for 1 hour at 37 ° C. followed by a fixed concentration (25 ng / ml) of IGF-1. After incubating the cells for 4 days at 37 ° C., proliferation was measured using the Promega Cell Titre Blue assay kit (Promega G8081). FIG. 41 shows dose-dependent inhibition of IGF-1-induced in vitro growth of NCI-H929 cells by H0L0 and parent 6E11.

Stability in Serum To study the stability of H0L0 in human serum, 500 ml aliquots of 100 μg / mL antibody were applied in human 100% serum at −20 ° C., 4 ° C. for up to 12 days. And incubated at 37 ° C. Activity after 12 days was measured by direct binding ELISA. EC50 values were calculated and compared to previous EC50 values of several previous binding ELISAs. The results shown in FIG. 42 below confirm that the activity of H0L0 does not decrease when incubated at −20 ° C., 4 ° C., or 37 ° C. for a period of up to 12 days in serum.

In order to confirm that the pharmacokinetics of receptor down-regulation H0L0 in xenograft models can down-regulate the IGF-1R receptor in vivo, an in vivo assay based on the LISN / 3T3 c4 cell line currently has other findings. It is under development (Cohen et al. (2005) Clin Cancer Res., 11 (5): 2063-73). In the first study, athymic CD1 nu / nu mice were transplanted with 2.5 × 10 ⁶ 3T3 / LISN cells in Matrigel (Becton Dickinson). When tumors reached 400-500 mm ³ in size, mice were administered 125 μg of control antibody or H0L0. Mice were sacrificed at T = 16, 24, 48, 72 or 120 hours after administration. Tumors were excised and immediately frozen in liquid nitrogen. Weighed tumor samples were homogenized in RIPA buffer + protease and phosphatase inhibitors and protein assays were performed on the lysates after centrifugation. A DELFIA assay (see Example 34) was performed to assess the relative levels of total IGF-1R in tumor samples. As shown in FIG. 43A, HOLO treatment appears to have some effect on all receptors in the 24-72 hour time frame, but the magnitude of the effect is greater than previously reported Is also quite small (Cohen, 2005).

In the second study, mice were transplanted (n = 6) as described above. When tumors reached a size of 400-500 mm ³ , mice were dosed with 250 μg of control antibody or H0L0 twice 72 hours apart. 24 hours after the final administration, 10 μg of human recombinant IGF-1 was administered intravenously. Ten minutes later, the tumor was excised and frozen in liquid nitrogen. A full IGF-1R DELFIA assay was performed as described in Example 34. Animals were treated with IGF-I, but no consistent changes were observed in phosphorylated receptor levels compared to the untreated control group. However, the group treated with anti-IGF-1R antibody (6E11, H0L0) showed a decrease at all receptor levels (FIG. 43B). Similar effects were seen in the last tumor sample in the efficacy study (data not shown). A combined study investigating receptor phosphorylation and effects over time on total receptor levels has proven inconclusive (data not shown).

100 μl of tumor lysate containing 10 or 25 μg of total IGF-1R DELFIA assay protein was loaded onto an ELISA plate pre-coated with anti-IGF-1R capture antibody 2B9 (see Example 13). The plates were then incubated overnight at 4 ° C. and washed with TBST. 100 μl of a 400 ng / ml polyclonal anti-IGF-1R biotin labeled antibody (R & D Systems BAF391) in 4% BSA / TBS was added to each well and incubated for 1 hour at room temperature. Plates were washed with TBST and 100 μl of 1/1000 dilution of Eu labeled streptavidin (Perkin Elmer, 1244-360) was added to each well and incubated for 1 hour at room temperature. The plate was washed with TBST and 100 μl of DELFIA enhancement solution (Perkin Elmer 1244-105) was added to each well and incubated for 10 minutes. Time-resolved fluorescence signals were measured using a Wallac Victor multilabel plate reader.

Sequence listing

SEQ ID NO: 1:
WILYYGRSKWYFDV

SEQ ID NO: 2:
NINPNNGGTNYNQKFKD

SEQ ID NO: 3
DYYMN

SEQ ID NO: 4:
RSSQSIVQSNGDTYLE

SEQ ID NO: 5
RISNRFS

SEQ ID NO: 6
FQGSHVPYT

SEQ ID NO: 7
RVSNRFS

SEQ ID NO: 8
EVQLQQSGPELVKPGASVRISCKASGYAFTDYYMNWVKQSHGKSLEWVANINPNNGGTNYNQKFKDKATLTVDKSSNTAYMELRSLTSEDTAVYYCARWILYYGRSKWYFDVWGTGTTVTVSS

SEQ ID NO: 9
DVLMTQTPLSLPVSLGDHASISCRSSQSIVQSNGDTYLEWYLQKPGQSPKLLIYRISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRA

SEQ ID NO: 10
QVQLKQSGPGLVQSSQSLSITCTISGFSLTSHGIYWLRQSPGKGLEWLGVIWSGGSADYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARSPYYYRSSLYAMDYWGQGTSVTVSS

SEQ ID NO: 11
NIVLTQSPKSMSMSIGERVTLSCKASENVGTYVSWYQQKAEQSPKLLIYGASNRHTGVPDRFTGSGSSTDFTLTISSVQAEDLADYHCGQSYSDPLTFGAGTKLELKRA

SEQ ID NO: 12
EVQLQQSGPELVKPGASVRISCKASGYAFTDYYMNWVKQSHGKSLEWVANINPNNGGTNYNQKFKDKATLTVDKSSNTAYMELRSLTSEDTAVYYCARWILYYGRSKWYFDVWGTGTLVTVSS

SEQ ID NO: 13
DVLMTQTPLSLPVSLGDHASISCRSSQSIVQSNGDTYLEWYLQKPGQSPKLLIYRISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRT

SEQ ID NO: 14
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSS

SEQ ID NO: 15
QVQLVQSGAEVKKPGASVKVSCKASGYAFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSS

SEQ ID NO: 16
DIVMTQSPLSLPVTPGEPASISCRSSQSIVQSNGDTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRT

SEQ ID NO: 17
GLNDIFEAQKIEWHE

SEQ ID NO: 18
EVQLQQSGPELVKPGASVRISCKASGYAFTDYYMNWVKQSHGKSLEWMANINPNNGGTNYNQKFKDKATLTVDKSSNTAYMELRSLTSEDSAVYYCARWILYYGRSKWYFDVWGPGTTVTVSS

SEQ ID NO: 19
DVLMTQSPLSLPVSLGDHASISCRSSQSIVQSNGDTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRA

SEQ ID NO: 20
EVQLQQSGPELVKPGASVKISCKASGYAFTDYYMNWVKQTHGRSLEWMANINPNTGGTNYNQKFRGKATLTVDKSSTTAYMELRSLTSEDSAVYYCARWILYYGSSRWYFDVWGTGTTVTVSS

SEQ ID NO: 21
DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPYTFGGGTKLEIKRA

SEQ ID NO: 22
EVQLQQSGPELVKPGASVKISCKASGYAFTDYYMNWVKQSHGKSLEWMANINPNTGGTNYNQKFTGKATLTVDKSSTTAYMELRSLTSEDSAVYYCTRWILYYGSSKWYFDVWGTGTTVTVSS

SEQ ID NO: 23
DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLLIYRVSYRFSGVPDRFSGSGSGTDFTLKISRLEAEDLGIYYCFQGSHVPYTFGGGTKLEIKRA

SEQ ID NO: 24
MGWSWIFFFLLSETAGVLSEVQLQQSGPELVKPGASVRISCKASGYAFTDYYMNWVKQSHGKSLEWVANINPNNGGTNYNQKFKDKATLTVDKSSNTAYMELRSLTSEDTAVYYCARWILYYGRSKWYFDVWGTGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 25
MKLPVRLVVLMFWIPASSSDVLMTQTPLSLPVSLGDHASISCRSSQSIVQSNGDTYLEWYLQKPGQSPKLLIYRISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTQVASGSVVQDSN

SEQ ID NO: 26:
GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGTAAGGCTTCTGGATACGCGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGGTGGCAAATATTAATCCCAACAATGGTGGTACTAACTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGATGGATTCTTTACTACGGTCGTAGCAAATGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCG

SEQ ID NO: 27:
GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCACGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGTTCAAAGTAATGGAGACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAGAATTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT

SEQ ID NO: 28:
GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGTAAGGCTTCTGGATACGCGTTCACTGACTACTACATGAACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAGTGGATGGCAAATATTAATCCCAACAATGGTGGTACTAACTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATGGATTCTTTACTACGGTCGTAGCAAGTGGTACTTCGATGTCTGGGGCCCAGGGACCACGGTCACCGTCTCCTCG

SEQ ID NO: 29
GATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCACGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTTCAAAGTAATGGAGACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTATAGAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT

SEQ ID NO: 30
GAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGTAAGGCTTCTGGATACGCGTTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGGTGGCAAATATTAATCCCAACAATGGTGGTACTAACTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGTCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGATGGATTCTTTACTACGGTCGTAGCAAATGGTACTTCGATGTCTGGGGCACAGGGACACTAGTCACAGTCTCCTCA

SEQ ID NO: 31
GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCACGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGTTCAAAGTAATGGAGACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAGAATTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACG

SEQ ID NO: 32


SEQ ID NO: 33:


SEQ ID NO: 34:
CAGGTCCAGCTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCCGGAGCTAGCGTCAAGGTCTCCTGCAAGGCTTCAGGCTACACATTCACCGACTACTACATGAACTGGGTGAGACAGGCTCCAGGACAGGGCCTCGAGTGGATGGGCAACATCAACCCCAACAATGGCGGGACAAACTACAACCAGAAGTTCAAGGATCGCGTGACCATGACCACCGACACTAGCACCTCAACAGCCTACATGGAGCTGAGGTCTCTGCGGAGCGATGACACTGCCGTGTACTACTGTGCCAGGTGGATTCTGTACTACGGGAGGAGCAAGTGGTACTTCGACGTCTGGGGAAGAGGGACACTAGTGACCGTGAGCAGC

SEQ ID NO: 35
CAGGTCCAGCTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCCGGAGCTAGCGTCAAGGTCTCCTGCAAGGCTTCAGGCTACGCCTTCACCGACTACTACATGAACTGGGTGAGACAGGCTCCAGGACAGGGCCTCGAGTGGATGGGCAACATCAACCCCAACAATGGCGGGACAAACTACAACCAGAAGTTCAAGGATCGCGTGACCATGACCACCGACACTAGCACCTCAACAGCCTACATGGAGCTGAGGTCTCTGCGGAGCGATGACACTGCCGTGTACTACTGTGCCAGGTGGATTCTGTACTACGGGAGGAGCAAGTGGTACTTCGACGTCTGGGGAAGAGGGACACTAGTGACCGTGAGCAGC

SEQ ID NO: 36
GACATCGTCATGACCCAGAGCCCACTGTCACTCCCCGTGACACCCGGAGAGCCCGCTAGCATCAGCTGTAGAAGCTCCCAGAGCATCGTGCAGTCTAACGGCGATACCTACCTCGAGTGGTACCTGCAGAAGCCCGGACAGTCTCCTCAGCTCCTGATTTACCGCGTCAGCAATCGCTTTTCCGGGGTGCCTGATCGGTTTAGCGGCTCAGGAAGCGGAACCGACTTCACCCTGAAGATCTCAAGGGTGGAGGCTGAGGATGTGGGCGTGTACTACTGCTTCCAGGGATCTCACGTGCCTTACACCTTCGGACAGGGCACAAAGCTCGAGATTAAGCGTACG

SEQ ID NO: 37:
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 38:
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYAFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 39:
MGWSCIILFLVATATGVHSDIVMTQSPLSLPVTPGEPASISCRSSQSIVQSNGDTYLEWYLQKPGQSPQLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPDSQTYLEQQQQQQ

SEQ ID NO: 40


SEQ ID NO: 41


SEQ ID NO: 42:


SEQ ID NO: 43
MGWSCIILFLVATATGVHS

SEQ ID NO: 44:


SEQ ID NO: 45:


SEQ ID NO: 46:


SEQ ID NO: 47:


SEQ ID NO: 48


SEQ ID NO: 49:
MGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

SEQ ID NO: 50:
MGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSAGLNDIFEAQKIEWHE

SEQ ID NO: 51:
MAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE

SEQ ID NO: 52:
MAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSEGLNDIFEAQKIEWHE

SEQ ID NO: 53:


SEQ ID NO: 54:
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 55:
ATGGGATGGTCCTGTATCATCCTGTTTCTGGTGGCCACAGCAACTGGCGTGCACTCTCAGGTCCAGCTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCCGGAGCTAGCGTCAAGGTCTCCTGCAAGGCTTCAGGCTACACATTCACCGACTACTACATGAACTGGGTGAGACAGGCTCCAGGACAGGGCCTCGAGTGGATGGGCAACATCAACCCCAACAATGGCGGGACAAACTACAACCAGAAGTTCAAGGATCGCGTGACCATGACCACCGACACTAGCACCTCAACAGCCTACATGGAGCTGAGGTCTCTGCGGAGCGATGACACTGCCGTGTACTACTGTGCCAGGTGGATTCTGTACTACGGGAGGAGCAAGTGGTACTTCGACGTCTGGGGAAGAGGGACACTAGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACAAGCGGGGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCTGAACTGGCCGGAGCCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAACAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGC TGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTGCCCCCCTCCCGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAGTGA

SEQ ID NO: 56:
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYAFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

SEQ ID NO: 57:


SEQ ID NO: 58:


SEQ ID NO: 59:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTXaaXaaXaaXaaXaaWVRQAPGQGLEWMGXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaRVTMTTDTSTSTAYMELRSLRSDDTAVYXCAXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa

SEQ ID NO: 60:
DIVMTQSPLSLPVTPGEPASISCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWYLQKPGQSPQLLIYXaaXaaXaaXaaXaaXaaXaaGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYXGTaaXaaXaaXaaXaaXaaXaaXaa

SEQ ID NO: 61:
CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAGGCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAGAAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACGGCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGT

SEQ ID NO: 62:
GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCTGGCGAGCCCGCCAGCATCAGCTGCAGAAGCAGCCAGAGCATCGTCCAGAGCAACGGCGACACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCTACAGAGTGAGCAACCGGTTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCCGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCTTTCAAGGCAGCCACGTGCCCTACACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGCGTACG

SEQ ID NO: 63


SEQ ID NO: 64
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 65


SEQ ID NO: 66
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 67


SEQ ID NO: 68
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 69


SEQ ID NO: 70
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAGGCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAGAAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACGGCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGC TGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGTGATGA

Claims (38)

  An antibody or antigen-binding fragment thereof that specifically binds to IGF-1R, comprising CDR H3 of SEQ ID NO: 1, or a variant thereof containing one or two amino acid substitutions in CDR H3.   The antibody or antigen-binding fragment according to claim 1, wherein the amino acid residues of SEQ ID NO: 1 differ by substitution at one or more positions selected from positions 7 and 9.   The antibody of claim 2, wherein the amino acid residues of SEQ ID NO: 1 differ by one or more substitutions selected from R to S substitution at position 7 and K to R substitution at position 9 Or an antigen-binding fragment.   The antibody or antigen-binding fragment has the following sequences: CDR H2 (SEQ ID NO: 2) or CDR H1 (SEQ ID NO: 3), CDR L1 (SEQ ID NO: 4), CDR L2 (SEQ ID NO: 7) and CDR L3 (SEQ ID NO: 6) The antibody or antigen-binding fragment according to any one of claims 1 to 3, further comprising one or more of:   5. The antibody or antigen-binding fragment of claim 4, wherein one or more CDRs may be replaced with a variant thereof, each variant CDR containing 1 or 2 amino acid substitutions.   6. The antibody or antigen-binding fragment according to claim 4 or 5, further comprising CDR H1 of SEQ ID NO: 3.   6. The antibody or antigen-binding fragment according to claim 4 or 5, further comprising CDR L2 of SEQ ID NO: 7. The following CDRs:
CDR H1: SEQ ID NO: 3
CDR H2: SEQ ID NO: 2
CDR H3: SEQ ID NO: 1
CDR L1: SEQ ID NO: 4
CDR L2: SEQ ID NO: 7
CDR L3: SEQ ID NO: 6
The antibody or antigen-binding fragment according to any one of claims 1 to 7, further comprising:   An antibody or antigen-binding fragment thereof, which comprises the heavy chain variable region of SEQ ID NO: 8 and the light chain variable region of SEQ ID NO: 9, and specifically binds to IGF-1R.   An antibody or antigen-binding fragment thereof, which comprises the heavy chain variable region of SEQ ID NO: 10 and the light chain variable region of SEQ ID NO: 11, and specifically binds to IGF-1R.   An antibody or antigen-binding fragment thereof, which comprises the heavy chain variable region of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 13, and specifically binds to IGF-1R.   An antibody or antigen-binding fragment thereof, which comprises the heavy chain variable region domain of SEQ ID NO: 14 and the light chain variable region domain of SEQ ID NO: 16, and specifically binds to IGF-1R.   An antibody or antigen-binding fragment thereof that comprises the heavy chain variable region of SEQ ID NO: 15 and the light chain variable region of SEQ ID NO: 16, and specifically binds to IGF-1R.   9. The CDR of any one of claims 1-8 or claim 9, wherein the antibody or antigen-binding fragment is rat, mouse, primate (eg, cynomolgus monkey, old world monkey or ape), or human. An antibody or antigen-binding fragment comprising the heavy or light chain variable region of any one of -13.   15. The antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody is a humanized antibody or a chimeric antibody.   16. The antibody or antigen binding fragment of any one of claims 1-15, wherein the antibody or antigen binding fragment further binds to primate IGF-1R.   The antibody or antigen-binding fragment according to any one of claims 1 to 16, wherein the antibody comprises a constant region.   18. The antibody of claim 17, wherein the antibody comprises a constant region of IgG isotype.   19. The antibody of claim 18, wherein the antibody is IgG1.   20. The antibody of any one of claims 1-19, comprising a constant domain region so that the ADCC and / or complement activation or effector function of the antibody is reduced.   20. The antibody according to any one of claims 1 to 19, comprising a mutated constant domain or a constant domain with a modified glycosylation profile such that the effector function / ADCC and / or complement activation of the antibody is enhanced. Antibodies. The fragment is Fab, Fab ′, F (ab ′) ₂ , Fv, diabody, triabody, tetrabody, miniantibody, minibody, isolated V _H or isolated _VL. The antigen-binding fragment according to any one of 1 to 17.   22. The antibody or claim of any one of claims 1-21, wherein the antibody or antigen-binding fragment thereof is capable of at least some effector function, eg, capable of some ADCC or CDC function. Item 23. The antigen-binding fragment according to any one of Items 1 to 22.   A recombinant transformed, transfected or transduced host cell comprising at least one expression cassette, said expression cassette comprising a heavy chain of an antibody or antigen-binding fragment according to the invention described herein. A host cell as described above, comprising a polynucleotide that encodes, and further comprising a polynucleotide that encodes the light chain of an antibody or antigen-binding fragment according to the invention described herein.   A recombinantly transformed, transfected or transduced host cell comprising at least one expression cassette, wherein the first expression cassette is a heavy chain of an antibody or antigen-binding fragment according to the invention described herein A host cell comprising a second expression cassette comprising a polynucleotide encoding a light chain of an antibody or antigen-binding fragment according to the invention as described herein.   26. The host cell according to claim 24 or 25, which is eukaryotic.   27. The host cell of claim 26, which is mammalian.   28. A host cell according to claim 27 which is CHO or NSO.   A method for producing an antibody according to any one of claims 1 to 21 or an antigen-binding fragment thereof according to any one of claims 1 to 22 in a serum-free culture medium. A method comprising culturing a host cell according to any one of the above.   30. The method of claim 29, wherein the antibody is secreted into the culture medium by the host cell.   31. The method of claim 30, wherein the antibody is further purified to at least 95% (eg, 98% or more) relative to the culture medium containing the antibody.   A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any of the preceding claims, and a pharmaceutically acceptable carrier.   35. A kit of parts comprising instructions for use with the composition of claim 32.   35. A method of treating a human patient suffering from cancer comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any of the preceding claims, or the composition of claim 32.   35. The method of claim 34, wherein the patient is suffering from breast cancer.   35. The method of claim 34, wherein the patient is suffering from prostate cancer.   Use of the antibody or antigen-binding fragment thereof according to any of the preceding claims in the manufacture of a medicament for treating a disease or disorder selected from the group consisting of rheumatoid arthritis, breast cancer, prostate cancer, lung cancer or myeloma.   The antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the antibody neutralizes the activity of IGF-IR.

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