monoclonal antibody, nucleotide sequence, expression vector, recombinant prokaryotic host cell, phar

专利摘要:
monoclonal antibody, nucleotide sequence, expression vector, host cell, pharmaceutical composition, use of the antibody, methods to inhibit the growth and / or proliferation of a tumor cell, to produce an antibody, to detect the presence of c-met in a sample, kit to detect the presence of c-met in a sample, and anti-idiotypic antibody. control device for a transmission mechanism, characterized by the fact that it is to control a stepped transmission mechanism (30), which includes a first clutch (32) to be engaged at the start, and a second clutch (33), different from the first clutch (32), and is locked when a hydraulic pressure is supplied to the first clutch (32) and the second clutch (33), and the first clutch (32) and the second clutch (33) are completely coupled, comprising: a hydraulic pressure control means (12) adapted to control a hydraulic pressure supplied to the stepped transmission mechanism (30), so that the first clutch (32) is placed in a fully coupled state, and the second clutch (33) is placed in a sliding locking state, in which the second clutch (33) is not fully engaged, in the case of a return from an inactive stop control, in which a motor (1) is automatically stopped. control device for a transmission mechanism according to claim 1, characterized by the fact that the hydraulic pressure control means (12) begins to reduce the hydraulic pressure, supplied to the second clutch (33), when an increased proportion of a speed of motor rotation per unit of time is less than a first predetermined value. control device for a transmission mechanism, according to claim 1 or 2, characterized by the fact that the hydraulic pressure control means (12) controls the hydraulic pressure, supplied to the second clutch (33), so that a decreased proportion of hydraulic pressure, supplied to the second clutch (33), per unit of time becomes a second predetermined value. control device for a transmission mechanism according to claim 1 or 2, characterized by the fact that the hydraulic pressure control means (12): includes the target output torque means (12), adapted for calculate a target output torque by adding a third predetermined value to a target output torque calculated last time; and controls the hydraulic pressure supplied to the second clutch (33), based on the target output torque calculated by means of calculating the target output torque (12). control device for a transmission mechanism, according to claim 1, characterized by the fact that the hydraulic control means (12); includes the means of calculating the target output torque (120, adapted to calculate an input torque of the stepped transmission mechanism, based on an engine rotation speed, and to calculate a target output torque of the stepped transmission mechanism (30 ), based on a deviation between the input torque of the stepped transmission mechanism (30), calculated at that time, and the input torque of the stepped transmission mechanism (30), calculated from the last time, and a four predetermined value; and controls the hydraulic pressure supplied to the second clutch (33), based on the target output torque calculated by means of calculating the target output torque (12). control device for a transmission mechanism, according to claim 5, characterized by the fact that the target output torque control means (12) calculates the target output torque, at that moment, by adding the deviation to the target output torque, calculated last time, when the svio is greater than the fourth predetermined value, or by adding the fourth predetermined value for the target output torque, calculated last time, when the deviation is less than the fourth predetermined value. control device for a transmission mechanism according to any one of claims 1 to 6, characterized in that it further comprises the gradient detection means (47), adapted to detect a gradient of a rolling track surface , in a direction of travel of a vehicle, where: an initial hydraulic pressure increases as an upward gradient of the surface of the roadway, in the direction of travel of the vehicle, increases. control device for a transmission mechanism according to any one of claims 1 to 7, characterized in that it further comprises the gradient detection means (47), adapted to detect a gradient of a bearing track surface , in a direction of travel of a vehicle, in which: the hydraulic pressure control means (12) causes the hydraulic pressure, supplied in the direction of incongruity (33), to be reduced from an initial hydraulic pressure, to a synchronization earlier, as an upward gradient of the track surface in the direction of travel of the vehicle increases. control device for a transmission mechanism according to any one of claims 1 to 8, characterized in that it further comprises the gradient detection means (47), adapted to detect a gradient of a rolling track surface , in a direction of travel of a vehicle, in which: the hydraulic pressure control means (12) reduces a decreased proportion of the hydraulic pressure, supplied to the second clutch (33), per unit of time, in the measure where a gradient upwards of the track surface, in the direction of travel of the vehicle, increases. control device for a transmission mechanism according to any one of claims 1 to 9, characterized in that the hydraulic pressure control means (12) initiates the supply of hydraulic pressure to the second clutch (33), from so that the second clutch (33) is adjusted in the sliding locking state when a vehicle stops. Control device for a transmission mechanism according to any one of claims 1 to 10, characterized in that the inactive brown control is initiated, after the second clutch (33) is adjusted in the sliding locking state. control method for a stepped transmission mechanism, which includes a first clutch (32), to be engaged at the start, and a second clutch (33), different from the first clutch (31), and which is locked when a hydraulic pressure is supplied to the first clutch (32) and the second clutch (33), and the first clutch (32) and the second clutch (33) are completely coupled, characterized by the fact that it comprises: control of a hydraulic pressure supplied to the transmission mechanism , so that the first clutch (32) is placed in a fully coupled state, and the second clutch (33) is placed in a sliding locking state, in which case the second clutch (33) is not fully engaged, in the case of a return from an inactive stop control, in which a motor (1) is automatically stopped.
公开号:BR112012022672B1
申请号:R112012022672
申请日:2011-03-10
公开日:2020-04-14
发明作者:Frank Labrijn Aran；De Goeij Bart；Van Den Brink Edward；Van De Winkel Jan；Schuurman Janine；J Neijssen Joost；Parren Paul；M A Hoet Rene
申请人:Genmab As；
IPC主号:

专利说明:

“MONOCLONAL ANTIBODY, NUCLEOTIDE SEQUENCE, EXPRESSION VECTOR, RECOMBINANT PROCARYIOTIC HOST CELL, PHARMACEUTICAL COMPOSITION, USE OF THE ANTIBODY, METHODS OF DETECTING THE PRESENCE OF ETHNICITY, IN THE PRESENCE OF ETHNICITY C-MET IN A SAMPLE ”
FIELD OF THE INVENTION [001] The present invention relates to monoclonal antibodies directed against human c-Met, the hepatocyte growth receptor, and the uses of such antibodies, in particular their use in the treatment of cancer.
BACKGROUND OF THE INVENTION c-Met is a membrane-crossing receptor tyrosine kinase protein. The mainly single chain precursor is cleaved after translation to produce the mature form of the c-Met heterodimer consisting of an extracellular α-chain (50 kDa) and a longer transmembrane β-chain (145 kDa), which are disulfide linked (Birchmeier et al. 2003. Nat. Rev. Mol. Cell. Biol. 4: 915). The extracellular part of c-Met is made up of three types of domains. The N-terminal SEMA domain is formed by the entire α-subunit and part of the β-subunit, and includes homology with the semaphorin proteins. The SEMA domain is followed by a domain rich in cysteine and then by four domains similar to immunoglobulin (Ig). The cytoplasmic part contains a justamembrane kinase domain and a carboxy-terminal tail which is essential for downstream signaling. The only known high-affinity ligand for c-Met, hepatocyte growth factor (HGF), is mainly expressed under normal conditions by fibroblasts (Li and Tseng 1995. J. Cell. Physiol.163: 61) and by tumor cells (Ferracini et al. 1995. Oncogene 10: 739). HGF (also called the dispersion factor, SF) is synthesized as a precursor that is proteolytically converted to a
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2/121 active α / β heterodimer. Based on the crystal structure of the receptor binding fragment, HGF is considered to bind to c-Met as a dimer (Chirgadze et al. 1999. Nat. Struct. Biol. 6:72). The HGF α-chain binds with high affinity in the Ig-like domain in c-Met, while the HGF β chain binds only with low affinity in the c-Met SEMA domain (Basilico et al. 2008. J. Biol, Chem. 283: 21267). The latter interaction is responsible for cMet dimerization and activation of receptor tyrosine kinase under binding of the active HGF heterodimer. Receptor autophosphorylation results in a unique contact site for effector recruitment, of which binding of Gab1 (binding protein 1 associated with the growth factor receptor bound to protein 2) is essential for the main signaling routes downstream of c-Met (Comoglio et al. 2008. Nat. Rev. Drug. Discov. 7: 504):
• Ras-ERK1 / 2 route: proliferation.
• Ras-Rac route: invasion, motility, epithelial-paramesenchymal transition.
• PI3K-Akt route: survival.
[002] c-Met is expressed on the surface of epithelial and endothelial cells of many organs during embryogenesis and in adulthood, including the liver, pancreas, prostate, kidneys, muscle, and bone marrow. Activation of c-Met plays an essential role in the so-called “invasive growth” program that consists of a series of processes, including proliferation, motility, angiogenesis and protection against apoptosis (Boccaccio and Comoglio 2006. Nat. Rev. Cancer 6: 637) . These c-Met-regulated processes occur under normal physiological conditions during embryonic development, repairs to liver and heart damage, and pathologically during oncogenesis (Eder et al. 2009. Clin. Cancer Res. 15: 2207).
[003] Inappropriate c-Met signaling occurs in virtually all types of solid tumors, such as bladder, breast, cervical,
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3/121 colorectal, gastric, head and neck, liver, lung, ovarian, pancreatic, prostate, renal, and thyroid, as well as in various sarcomas, hematopoietic malignancies and melanoma (Birchmeier et al. 2003. Nat. Rev. Mol. Cell Biol. 4: 915; Comoglio et al. 2008. Nat. Rev. Drug Discov. 7: 504; Peruzzi and Bottaro 2006. Clin. Cancer Res. 12: 3657). The mechanisms underlying c-Met tumorigenicity are typically performed in three ways:
• autocrine HGF / c-Met loops, • overexpression of c-Met or HGF, • kinase-activating mutations in the c-Met receptor coding sequence.
[004] Most notably, c-Met activating mutations have been identified in patients with hereditary papillary kidney cancer (Schmidt et al. 1997. Nat. Genet. 16:68). Constitutive activation of c-Met contributes to one or more proliferative, invasive, surviving, or angiogenic cancer phenotypes. It has been shown that silencing of c-Met gene endogenously expressed in tumor cells results in lack of tumor proliferation and growth and regression of established metastasis, as well as decreased generation of new metastases (Corso et al. 2008. Oncogene 27: 684).
[005] Since c-Met contributes to multiple stages of cancer development, from initiation to progression to metastasis, c-Met and its HGF ligand have become leading candidates for selected cancer therapies (Comoglio et al. 2008. Nat. Rev. Drug. Discov. 7: 504; Knudsen and Vande Woude 2008. Curr. Opin. Genet. Dev. 18:87). Several strategies are being explored to achieve this goal:
• Decoy receptors: sub-regions of HGF or c-Met or molecular analogs can act antagonistically as stoichiometric competitors by blocking ligand binding or receptor dimerization. An example of an antagonistic sub-region of HGF is NK4
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4/121 (Kringle Pharma).
• Small molecule tyrosine kinase inhibitors (TKIs): Three c-Met specific TKIs at different stages of clinical evaluation are ARQ197 (ArQule), JNJ 38877605 (Johnson & Johnson) and PF-04217903 (Pfizer).
• Anti-HGF monoclonal antibodies, such as AMG102, rilotumumab (Amgen), HuL2G7 (Takeda), and AV-299 (Schering).
• Anti-c-Met monoclonal antibodies have been described in
WO2005016382, WO2006015371, WO2007090807, WO2007126799
WO2009007427, WO2009142738 and van der Horst et al. (van der Horst et al. 2009. Neoplasia 11: 355). MetMAb (Genentech) is a humanized monovalent (one-armed) OA-5D5 antibody that binds to the extracellular domain of cMet, thereby preventing HGF binding and subsequent receptor activation (Jin et al. 2008. Cancer Res. 68 : 4360). In mouse xenograft models, treatment with MetMAb has been found to inhibit the growth of HGF-activated orthotopic glioblastoma tumor and subcutaneous pancreatic tumors (Jin et al. 2008. Cancer Res. 68: 4360; Martens et al. 2006. Clinens Cancer Res. 12: 6144). h224G11 (Pierre Fabre) (Boute Corvaia and 2009. Abstract 835 100 AACR Annual Meeting) is a bivalent humanized IgG anti-c-Met. Antitumor effects of this antibody have been observed in mice (Goetsch et al. 2009. Abstract 2792 AACR 100 ^o Annual Meeting). CE-355621 (Pfizer) is a human IgG2 that blocks ligand binding by binding in the extracellular domain of c-Met and inhibits HGF-dependent growth in tumor xenograft models (Tseng et al. 2008. J. Nucl. Med. 49: 129).
[006] In conclusion, several anti-c-Met products are under investigation, but so far no product has been approved for therapeutic use. There remains a need for effective and safe products to treat c-Met-related illnesses, such as cancer.
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SUMMARY OF THE INVENTION [007] An object of the present invention is to provide highly specific and effective anti-c-Met monoclonal antibodies for medical use. The antibodies of the invention exhibit c-Met binding characteristics that differ from the antibodies described in the art. In preferred embodiments, the antibodies of the invention have a high affinity for human c-Met, are antagonistic and have a favorable pharmacokinetic profile for use in human patients.
BRIEF DESCRIPTION OF THE DRAWINGS [008] Figure 1: Alignment of HuMabs heavy chain variable region sequences. Based on these sequences, a consensus sequence can be defined for some of the CDR sequences. These consensus strings are shown in Table 4.
[009] Figure 2: Alignment of HuMabs light chain variable region sequences. Based on these sequences, a consensus sequence can be defined for some of the CDR sequences. These consensus strings are shown in Table 4.
[0010] Figure 3: Binding curves of monovalent and divalent forms of anti-c-Met antibodies to A431 cells expressing c-Met. Data shown is MFI from a representative experiment. Due to the fact that IgG1-024 and Uni-068 did not show saturated binding in A431 cells, it was not possible to calculate an accurate EC50 value.
[0011] Figure 4: Binding of antibodies to c-Met expressed in Rhesus monkey epithelial cells. Data shown is MFI from an experiment.
[0012] Figure 5: Anti-c-Met antibody-induced inhibition of HGF binding in the extracellular domain of the c-Met receptor. Data shown is a representative experiment.
[0013] Figure 6: HGF binding inhibition curves of the various
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6/121 anti-c-Met antibodies for binding to cMetSEMA_567His8 tested with TRFRET. Data shown are mean MFI ± standard deviation of three independent experiments.
[0014] Figure 7: Percent inhibition of viable KP4 cells after treatment with anti-c-Met antibody compared to untreated cells (0%). Data shown are percentages of inhibition of viable cells from two independent experiments ± the standard deviation. IgG1-1016-022 was only positive in one experiment.
[0015] Figure 8: Efficacy of anti-c-Met antibodies to inhibit tumor growth in a KP4 xenograft model in SCID mice. Mice were treated with 400 gg of antibody on day 9 followed weekly by a maintenance dose of 200 gg. Average tumor sizes by treatment group are shown.
[0016] Figure 9: Efficacy of anti-c-Met antibodies to inhibit tumor growth in a KP4 xenograft model in SCID mice. Mice were treated with 400 gg of antibody on day 9 followed weekly by a maintenance dose of 200 gg. Treatment effect on tumor incidence over time. The percentage of tumor-free mice (tumor sizes <500 mm3) is shown. Tumor formation is delayed in mice treated with antagonistic antibodies compared to control antibodies.
[0017] Figure 10: Efficacy of anti-c-Met antibodies to inhibit tumor growth in a MKN45 xenograft model in SCID mice. Mice were treated with 40 mg / kg of antibody on the 7 and 20 mg / kg of antibody on days 14, 21 and 28. Average tumor sizes are shown up to 50% of the mice reached 700 mm ³ endpoint, per group of treatment.
[0018] Figure 11: Efficacy of anti-c-Met antibodies to inhibit tumor growth in a MKN45 xenograft model in
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7/121 SCID mice. Mice were treated with 40 mg / kg of antibody at 7 and 20 mg / kg of antibody on days 14, 21 and 28. Percentage of mice with tumor sizes less than 700 mm3 is shown on a Kaplan Meier plot. Tumor formation is delayed in mice treated with anti-c-Met antibodies compared to the isotype control antibody.
[0019] Figure 12: KP4 viability assay to determine the effect of antibody flexibility on antagonistic activity. The IgA2m (1) format did not induce proliferation, unlike the IgA1 and IgG1 formats of the same antibody. Variants of the anti-c-Met 5D5 antibody (see US6468529 and Example 2) were used in this treatment.
[0020] Figure 13: Analysis by non-reduced SDS-PAGE of (069) flexibility mutants. No aberrant multimers or degradation products were observed, while light chain matching was visible as a 50 kD band ((LQ2) in mutants C220S, AC220 and IgG1 IgG3 articulation.
[0021] Figure 14: Antigen binding ELISA to measure the c-Met binding of anti-c-Met antibody joint mutants. All mutants bind with comparable affinity in c-Met as shown in ELISA.
[0022] Figure 15: C-Met phosphorylation as a reading for agonistic activity of antibodies against c-Met. Figure 15 shows Western blot results of lipases A549; membranes stained with antibodies against phosphorylated cMet, total c-Met or β-actin.
[0023] Figure 16: Proliferation assay with NCI-H441 cells. Cell mass was determined after 7 days of incubation in the presence of the antibody or controls and plotted as a percentage of untreated samples (attributed to 100%).
[0024] Figure 17: KP4 viability test. The effect was tested
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8/121 of antibodies against c-Met on the total viability of KP4 cells. The ability of IgG1-1016-069 to reduce the viability of KP4 has been retained and / or improved by introducing mutations that decrease the flexibility of the antibodies.
[0025] Figure 18: Inframodulation as measured as total levels of c-Met in A549 lysates using ELISA. All antibody variants (069) retained the ability to inframodulate.
[0026] Figure 19: ADCC assay to compare high and low fucose versions of the IgG1-1016-069 antibody.
[0027] Figure 20: Lack of binding of anti-c-Met antibodies in cells in whole blood in binding assay using FACS (fluorescence activated cell fractionator). The results are shown for B cells; monocytes and granulocytes.
DETAILED DESCRIPTION OF THE INVENTION
Definitions [0028] The term “c-Met”, when used herein, refers to the hepatocyte growth factor receptor (Gene Bank Accession Number NM 000245) and includes any variants, isoforms and homologs of species of c -Met from human that are naturally expressed by cells or are expressed on cells transfected with the c-Met gene.
[0029] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light chains (L) of low molecular weight and one pair of heavy chains (H), all the four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for example “Fundamental Immunology” Ch. 7 (Paul, W., ed., 2 ^nd ed. Raven Press, New York (1989)). Briefly, each heavy chain is typically comprised of a heavy chain variable region (here abbreviated as VH or VH) and a heavy chain constant region. The region
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9/121 heavy chain constant is typically comprised of three domains, Ch1, Ch2, and Ch3. Each light chain is typically comprised of a light chain variable region (here abbreviated as V1 or VL) and a light chain constant region. The light chain constant region is typically comprised of a domain, Cl. The Vh and Vl regions can be further divided into regions of hypervariability (or hypervariable regions that can be hypervariable in sequence and / or structurally defined loop shapes), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called mold regions (FRs). Each Vh and Vl is typically composed of three CDRs and four FRs, positioned from the amino termination to the carboxyl termination in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol Biol. 196. 901-917 (1987)). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (phrases such as variable domain residue numbering as in Kabat or according to Kabat refer here to this numbering system for heavy chain variable domains or light chain variable domains). By using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer amino acids or additional amino acids corresponding to a shortening of, or insertion into, a variable domain FR or CDR. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of Vh CDR2 and inserted residues (for example, residues 82a, 82b, and 82c, etc. according with Kabat) after residue 82 of heavy chain FR. The Kabat numbering of residues can be determined by a given antibody by aligning in regions of homology of the
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10/121 antibody with a "standard" Kabat numbered sequence.
[0030] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any one of them, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about an hour, at least about two hours , at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days , etc., or any other relevant functionally defined period (such as sufficient time to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or sufficient time for the antibody to recruit an effector activity ). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. Antibody constant regions (Abs) can mediate immunoglobulin binding with host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classic route of activation of complement. An anti-Met antibody can also be a bispecific antibody, diabody, or similar molecule (see, for example, PNAS USA 90 (14), 6444-8 (1993) for a description of diabody). In fact, bispecific antibodies, diabodies, and the like, provided by the present invention can bind to any suitable target in addition to a portion of c-Met. As indicated above, the term antibody here, unless otherwise indicated or clearly contradicted by the context, includes fragments of an antibody that retain the ability to specifically bind to the antigen. It has been shown that the
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11/121 Antigen binding of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antibody" include (i) a Fab 'or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) F (ab ') 2 fragments, divalent fragments comprising two Fab fragments linked by a disulfide bridge in the articulation region; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341. 544-546 (1989)), which consists essentially of a Vh domain and also called domain antibodies (Holt et al .; Trends Biotechnol. 2003 Nov; 21 (11): 484-90); (vi) antibodies to camelids or nanobodies (Revets et al .; Expert Opin. Biol. Ther. 2005 Jan; 5 (1): 111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two Fv fragment domains, VL and VH, are encoded by two separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be prepared as a single protein chain in which the regions VL and VH pair to form monovalent molecules (known as single chain antibodies or single chain Fv (Fv, scFv), see, for example, Bird et al., Science 242. 423426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are included within the term antibody unless otherwise indicated or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other antibody fragments useful in the context of the present invention are discussed hereinafter. It should also be understood that the term
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12/121 antibody, unless otherwise indicated, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (binding fragments) antigen) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can have any isotype. [0031] As used herein, "isotype" refers to the class of immunoglobulin (for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by the heavy chain constant region genes.
[0032] The term "monovalent antibody" means in the context of the present invention that an antibody molecule is capable of binding to a single antigen molecule, and thus is not capable of cross-linking antigens.
[0033] An "antibody deficient in effector function" or an "antibody that is deficient in effector function" refers to an antibody that has a significantly reduced ability, or no ability, to activate one or more effector mechanisms, such as activation complement or Fc receptor binding. Thus, antibodies deficient in effector function have significantly reduced or no capacity to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). An example of such an antibody is IgG4.
[0034] An "anti-c-Met antibody" is an antibody as described above, which specifically binds to the c-Met antigen.
[0035] The term "human antibody", as used herein, is intended to include antibodies having constant and variable regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can include residues of
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13/121 amino acid not encoded by human germline immunoglobulin sequences (eg, mutations introduced by site-specific or random mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been graftized into sequences from the mold region of human.
[0036] As used herein, a human antibody is "derived from" a special germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying immunoglobulin genes from human or by screening a human immunoglobulin gene library, and the selected human antibody is at least 90%, such as at least 95%, for example, at least 96%, such as at least 97% , for example, at least 98%, or as at least 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, outside of the heavy chain CDR3, a human antibody derived from a special human germline sequence will exhibit no more than 20 different amino acids, e.g. , no more than 10 different amino acids, such as no more than 9, 8, 7, 6 or 5, for example, no more than 4, 3, 2, or 1 different amino acid (s) of the amino acid sequence encoded by the germline immunoglobulin gene.
[0037] In a preferred embodiment, the antibody of the invention is isolated. An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to c-Met is substantially free of
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14/121 antibodies that specifically bind to antigens other than c-Met). An isolated antibody that specifically binds to an epitope, an isoform or a variant of c-Met may, however, cross-react with other related antigens, for example, from other species (such as c-Met species homologs). In addition, an isolated antibody can be substantially free of other chemical compounds and / or cellular materials. In one embodiment of the present invention, two or more "isolated" monoclonal antibodies having different antigen binding specificities are combined into a well-defined composition.
[0038] When used here in the context of two or more antibodies, the term "competes with" or "competes crosswise with" indicates that the two or more antibodies compete for binding in c-Met, p. , compete for c-Met binding in the assay described here in the Examples. For some pairs of antibodies, competition in the Examples assay is only observed when one antibody is coated on the plate and the other is used to compete, and not vice versa. The term "competes with" when used here is also intended to cover such combinations of antibodies.
[0039] The term "epitope" means a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of surface clusters of molecules such as amino acids or sugar side chains and usually specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that the binding of the former, but not the latter, is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in binding, such as amino acid residues that are effectively blocked by the antigen specific binding peptide ( in
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Other words, the amino acid residue is within the range of action of the antigen specific ligand peptide).
[0040] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of unique molecular composition. A monoclonal antibody composition exhibits a unique binding specificity and affinity for a special epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies exhibiting a unique binding specificity that has constant and variable regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be generated by a hybridoma that includes a B cell obtained from a transgenic or transromosomal non-human animal, such as a transgenic mouse, having a genome comprising a heavy chain transgene and a light chain transgene, fused in an immortalized cell.
[0041] As used herein, the term "binding" in the context of binding an antibody to a predetermined antigen is typically a binding with an affinity corresponding to a Kd of about 10 ^-7 M or less, such as about 10 ^{- 8} M or less, such as about 10 ^-9 M or less, about 10 ^-10 M or less, or about 10 ^-11 M or even less when determined, for example, by surface plasmon resonance technology ( SPR) on a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and bonds on the predetermined antigen with an affinity corresponding to a KD that is at least ten times less, such as at least 100 times less, for example at least 1,000 times less, such as at least 10,000 times less, for example, at least 100,000 times less than its affinity for binding to a non-specific antigen (eg, BSA, casein) other than the predetermined antigen or a closely related antigen O. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody
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16/121 antibody is very low (that is, the antibody is highly specific), so the amount with which the affinity for the antigen is less than the affinity for a non-specific antigen can be at least 10,000 times.
[0042] The term "kd" (s ^-1 ), as used here, refers to the dissociation rate constant of a special antibody-antigen interaction. Said value is also called the koff value.
[0043] The term "ka" (M ^-1 xs ^-1 ), as used here, refers to the rate of association of a special antigen-antibody interaction.
[0044] The term “KD” (M), as used here, refers to the dissociation equilibrium constant of a special antibody-antigen interaction.
[0045] The term "Ka" (M ^-1 ), as used here, refers to the equilibrium constant constant of a special antibody-antigen interaction and is obtained by dividing ka by kd.
[0046] As used herein, the term "inhibits growth" (eg, referring to cells, such as tumor cells) is intended to include any measurable decrease in cell growth when contacted with an anti- c-Met compared to the growth of the same cells not in contact with an anti-c-Met antibody, e.g. e.g., the growth inhibition of a cell culture is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100 %. Such a decrease in cell growth can occur by a variety of mechanisms, e.g. effector cell phagocytosis, ADCC, CDC, and / or apoptosis. [0047] The present invention also provides antibodies comprising functional variants of the VL region, the VH region, or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of an anti-c-Met antibody still allows the antibody to retain at least a substantial proportion (at least
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17/121 about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or specificity / selectivity of the parental antibody and in some cases an anti-c-Met antibody may be associated with greater affinity, selectivity and / or specificity than that of the parental antibody.
Such functional variants typically retain significant sequence identity with the parental antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, homology% = [number of identical positions / total number of positions] x 100), considering microorganism number of gaps, the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide sequences or amino acid sequences can be e.g. eg, determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a weight residual table PAM120, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage identity between the two amino acid sequences can be determined using the Needleman and Wunsch algorithm, J. Mol. Biol. 48, 444-453 (1970).
[0049] The sequence of CDR variants may differ from the CDR sequence of parental antibody sequences by means of mostly conservative substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue substitutions.
[0050] In the context of the present invention, conservative substitutions can be defined by substitutions within the classes of amino acids reflected in the following table:
Classes of amino acid residues for conservative substitutions Acid Waste Asp (D) and Glu (E)
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Basic Waste Lys (K), Arg (R), and His (H) Unloaded Hydrophilic Waste Ser (S), Thr (T), Asn (N), and Gln (Q) Unloaded Aliphatic Waste Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Non-Loaded Waste Cys (C), Met (M), and Pro (P) Aromatic Waste Phe (F), Tyr (Y), and Trp (W)
[0051] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced, e.g. an expression vector encoding an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS / 0 cells, and lymphocytic cells.
[0052] The term "transgenic non-human animal" refers to a non-human animal having a genome comprising one or more transchromosomes or human heavy and / or light chain transgenes (whether integrated or not integrated into the animal's natural genomic DNA ) and that is capable of expressing fully human antibodies. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse produces human anti-c-Met antibodies when immunized with c-Met antigen and / or cells expressing c-Met. The human heavy chain transgene can be integrated into the mouse chromosomal DNA, as is the case for transgenic mice, such as HCo7 or HCo12 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for mice Extrachromosomal KM as described in WO02 / 43478. Similar mice, having a larger human Ab gene repertoire, include HCo7 and HCo20 (see, eg, WO2009097006). Such transgenic and trans-chromosomal mice (collectively referred to here as “transgenic mice”) are capable of producing multiple isotypes of human monoclonal antibodies to a given antigen (such as IgG,
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IgA, IgM, IgD and / or IgE) by subjecting the V-D-J recombination and isotype switching. Transgenic, non-human animal can also be used to produce antibodies against a specific antigen by introducing genes encoding that specific antibody, for example by operatively linking the genes to a gene that is expressed in the animal's milk.
[0053] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of facilitating, ameliorating, interrupting or eradicating (curing) sick symptoms or conditions.
[0054] An "effective amount" refers to an effective amount, in dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an anti-c-Met antibody can vary according to factors such as the individual's sickness, age, sex, and weight, and the ability of the anti-c-Met antibody to produce a response desired in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the antibody or portion of the antibody are outweighed by the therapeutically beneficial effects.
[0055] An "anti-idiotypic" antibody is an antibody that recognizes unique determinants generally associated with an antibody's antigen binding site.
Other aspects and other embodiments of the invention [0056] As described above, in a first aspect, the invention relates to a monoclonal antibody that binds to a human c-Met.
[0057] Monoclonal antibodies of the present invention can be e.g. , produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or can be produced by recombinant DNA methods. Monoclonal antibodies can also be isolated from antibody phage libraries using the techniques described, for example,
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20/121 in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for example in the form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen from interest. Monoclonal antibodies can also be obtained from hybridomas derived from cells expressing antibody from immunized human or non-human mammals such as rats, dogs, primates, etc.
[0058] In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies produced against c-Met can be generated using transgenic or trans-chromosomal mice transporting parts of the human immune system instead of the mouse immune system. Such transgenic or transchromosomal mice include mice here called HuMAb mice and KM mice, respectively, and are collectively referred to here as "transgenic mice".
[0059] The HuMAb mouse contains a human immunoglobulin gene minilocus that encodes non-rearranged human κ light chain immunoglobulin and heavy chains (μ and γ), along with selected mutations that inactivate the μ and κ chain loci endogenous (Lonberg, N. et al., Nature 368. 856-859 (1994)). Consequently, mice exhibit reduced expression of mouse IgM or κ in response to immunization, introduced human light and heavy chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG, K monoclonal antibodies (Lonberg , N. et al. (1994), supra; revised in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D.,
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Intern. Rev. Immunol. Vol. 65-93 (1995) and Harding, F. and Lonberg, N. Ann. New York. Acad. Sci. 764 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al. , Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770. 429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
[0060] HCo7 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their chain genes heavy proteins (as described in Example 1 of WO 01/14424), a human KCo5 kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and a chain transgene heavy human HCo7 (as described in US 5,770,429).
[0061] HCo12 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their chain genes heavy proteins (as described in Example 1 of WO 01/14424), a human KCo5 kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and a chain transgene human heavy weight HCo12 (as described in Example 2 of WO 01/14424).
[0062] In the KM mouse strain, the endogenous mouse kappa light chain gene has been subjected to homozygous disruption as described in Chen et al., EMBO J. 12, 811-820 (1993) and the heavy chain gene of endogenous mice have been subjected to homozygous disruption as described in Example 1 of WO 01/09187. This strain of
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22/121 mouse carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 hCF fragment (SC20) as described in WO 02/43478.
[0063] Splenocytes from these transgenic mice can be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.
[0064] Furthermore, antibodies of human of the present invention or antibodies of the present invention of other species can be identified by means of display-type technology, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to further maturation, such as affinity maturation, as such techniques are known in the art (see for example Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech. 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids Research 21, 10811085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBT ECH 10, 80-84 (1992), and US 5,733,743). If display technologies are used to produce non-human antibodies, such antibodies can be humanized.
[0065] In one embodiment, the antibody of the invention is of the IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM isotype.
[0066] In a first major embodiment of the antibody of the invention, the antibody competes to bind soluble cMetECDHis with an immobilized antibody, said immobilized antibody comprising a
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23/121 VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 37 (024), preferably the antibody competing in more than 50%, such as more than 75 % with said immobilized antibody, when determined as described in Example 17.
[0067] In yet another embodiment, the antibody does not compete to bind soluble cMetECDHis with an antibody selected from the group consisting of:
a) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 5 (005)
b) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 21 (008)
c) an immobilized antibody comprising the VH region and the VL region of antibody 5D5, and
d) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 53 (045), preferably the antibody competing in less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
[0068] In yet another embodiment, the antibody binds to the same epitope as that of an antibody selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 37 (024)
b) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 65 and a VL region
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24/121 comprising the sequence of SEQ ID NO: 69 (061)
c) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 73 and a VL region comprising the sequence of SEQ ID NO: 77 (062)
d) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 81 and a VL region comprising the sequence of SEQ ID NO: 85 (064)
e) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 89 and a VL region comprising the sequence of SEQ ID NO: 93 (068)
f) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 97 and a VL region comprising the sequence of SEQ ID NO: 101 (069)
g) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 113 and a VL region comprising the sequence of SEQ ID NO: 117 (098)
h) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 121 and a VL region comprising the sequence of SEQ ID NO: 125 (101), and
i) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 129 and a VL region comprising the sequence of SEQ ID NO: 133 (181).
[0069] In yet another embodiment, the antibody comprises a VH CDR3 region having the sequence as shown in
a) SEQ ID NO: 36 (024)
b) SEQ ID NO: 193, such as a VH CDR3 region as shown in SEQ ID NO: 68, 76, 84 or 92 (061, 062, 064, 068)
c) SEQ ID NO: 196, such as a VH CDR3 region as shown in SEQ ID NO: 100 or 132 (069, 181)
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d) SEQ ID NO: 116 (098), or
e) SEQ ID NO: 201, such as a VH CDR3 region as shown in SEQ ID NO: 124 (101).
[0070] In yet another embodiment, the antibody comprises:
a) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 34, 185 and 36 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 38, 39 and 206, as an antibody comprising a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 34, 35 and 36 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 38, 39 and 40, ( 024)
b) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 191, 192 and 193 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 78, 79 and 208, as an antibody comprising
The. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 66, 67 and 68 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 70, 71 and 72 (061)
B. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 74, 75 and 76 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 78, 79 and 80, (062)
ç. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 82, 83 and 84 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 86, 87 and 88, (064), or
d. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 90, 91 and 92 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 94, 95 and 96, (068)
c) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 194, 195 and 196 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 209, 210 and 104, as one
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26/121 antibody comprising
The. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 98, 99 and 100 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 102, 103 and 104, (069), or
B. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 130, 131 and 132 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 134, 135 and 136, (181)
d) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 197, 198 and 116 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 118, 119 and 211, as an antibody comprising a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 114, 115 and 116 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 118, 119 and 120 (098 ), or
e) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 199, 200 and 201 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 126, 212 and 128, as an antibody comprising a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 122, 123 and 124 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 126, 127 and 128 (101 ).
[0071] In yet another embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO: 33 and, preferably, a VL region comprising the sequence of SEQ ID NO: 37 (024)
b) a VH region comprising the sequence of SEQ ID NO: 61 and, preferably, a VL region comprising the sequence of SEQ ID NO: 69 (061)
c) a VH region comprising the sequence of SEQ ID
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NO: 73 and, preferably, a VL region comprising the sequence of SEQ ID NO: 77 (062)
d) a VH region comprising the sequence of SEQ ID NO: 81 and, preferably, a VL region comprising the sequence of SEQ ID NO: 85 (064)
e) a VH region comprising the sequence of SEQ ID NO: 89 and, preferably, a VL region comprising the sequence of SEQ ID NO: 93 (068)
f) a VH region comprising the sequence of SEQ ID NO:
and, preferably, a VL region comprising the sequence of SEQ ID NO: 101 (069)
g) a VH region comprising the sequence of SEQ ID NO: 113 and, preferably, a VL region comprising the sequence of SEQ ID NO: 117 (098)
h) a VH region comprising the sequence of SEQ ID NO: 121 and, preferably, a VL region comprising the sequence of SEQ ID NO: 125 (101)
i) a VH region comprising the sequence of SEQ ID NO:
129 and, preferably, a VL region comprising the sequence of SEQ ID NO: 133 (181)
j) a VH region comprising the sequence of SEQ ID NO:
159 and preferably a VL region comprising the sequence of SEQ ID NO: 160 (078)
k) a VH region comprising the sequence of SEQ ID NO: 161 and, preferably, a VL region comprising the sequence of SEQ ID NO: 162 (084)
l) a VH region comprising the sequence of SEQ ID NO:
163 and preferably a VL region comprising the sequence of SEQ ID NO: 164 (063)
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m) a VH region comprising the sequence of SEQ ID NO: 165 and, preferably, a VL region comprising the sequence of SEQ ID NO: 166 (087)
n) a VH region comprising the sequence of SEQ ID NO: 137 and, preferably, a VL region comprising the sequence of SEQ ID NO: 138 (066)
o) a VH region comprising the sequence of SEQ ID NO: 139 and, preferably, a VL region comprising the sequence of SEQ ID NO: 140 (065)
p) a VH region comprising the sequence of SEQ ID NO: 141 and, preferably, a VL region comprising the sequence of SEQ ID NO: 142 (082)
q) a VH region comprising the sequence of SEQ ID NO: 143 and, preferably, a VL region comprising the sequence of SEQ ID NO: 144 (089), or
r) a variant of any one of said antibodies, said variant preferably having at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.
[0072] In one embodiment, the antibody comprises a VH region comprising the CDR3 sequence of SEQ ID NO: 100 and a VL region comprising the CDR3 sequence of SEQ ID NO: 104, (069).
[0073] In one embodiment, the antibody comprises a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 98, 99 and 100 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 102, 103 and 104, (069).
[0074] In one embodiment, the antibody comprises a VH region comprising the sequence of SEQ ID NO: 97 and a VL region comprising the sequence of SEQ ID NO: 101 (069).
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29/121 [0075] In another main embodiment of the antibody of the invention:
- the antibody competes to bind soluble cMetECDHis with an immobilized antibody, said immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 13 (006), preferably the antibody competing in more than 50%, such as more than 75% with said immobilized antibody, when determined as described in Example 17, and
- the antibody does not compete to bind to soluble cMetECDHis with an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 53 (045), preferably with the antibody competing less than 50%, e.g. less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17 and
- the antibody binds in the SEMA domain of c-Met, preferably the antibody being able to inhibit HGF binding in the SEMA domain with an IC 50 of less than 10 gg / ml, such as less than 2 gg / ml mL as described in Example 9.
[0076] In yet another embodiment, the antibody does not compete to bind soluble cMetECDHis with an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 37 (024) , preferably with the antibody competing in less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
[0077] In yet another embodiment, the antibody binds to the same epitope as that of an antibody selected from the group consisting of:
a) an antibody comprising a VH region
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30/121 comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 5 (005)
b) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 13 (006)
c) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 29 (022), and
d) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 57 and a VL region comprising the sequence of SEQ ID NO: 61 (058).
[0078] In yet another embodiment, the antibody comprises a VH CDR3 region having the sequence as shown in
a) SEQ ID NO: 181, such as a VH CDR3 region as shown in SEQ ID NO: 4 or 12 (005, 006)
b) SEQ ID NO: 28 (022), or
c) SEQ ID NO: 60 (058).
[0079] In yet another embodiment, the antibody comprises:
a) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 179, 180 and 181 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 6, 7 and 202, as an antibody comprising
The. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 2, 3 and 4 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 6, 7 and 8, (005), or
B. a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 10, 11 and 12 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 14, 15 and 16, (006)
b) a VH region comprising the sequences of CDR1, 2 and
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31/121 of SEQ ID NO: 26, 184 and 28 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 30, 31 and 205, such as an antibody comprising a VH region comprising the sequences of CDR1 , 2 and 3 of SEQ ID NO: 26, 27 and 28 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 30, 31 and 32 (022), or
c) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 189, 190 and 60 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 62, 63 and 207, as an antibody comprising a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 58, 59 and 60 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 62, 63 and 64 (058 ) [0080] In yet another embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO: 1 and, preferably, a VL region comprising the sequence of SEQ ID NO: 5 (005)
b) a VH region comprising the sequence of SEQ ID NO: 9 and, preferably, a VL region comprising the sequence of SEQ ID NO: 13 (006)
c) a VH region comprising the sequence of SEQ ID NO: 25 and, preferably, a VL region comprising the sequence of SEQ ID NO: 29 (022)
d) a VH region comprising the sequence of SEQ ID NO: 57 and, preferably, a VL region comprising the sequence of SEQ ID NO: 61 (058)
e) a VH region comprising the sequence of SEQ ID NO: 145 and, preferably, a VL region comprising the sequence of SEQ ID NO: 146 (031)
f) a VH region comprising the sequence of SEQ ID NO:
147 and preferably a VL region comprising the SEQ sequence
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ID NO: 148 (007)
g) a VH region comprising the sequence of SEQ ID NO: 149 and, preferably, a VL region comprising the sequence of SEQ ID NO: 150 (011)
h) a VH region comprising the sequence of SEQ ID NO: 151 and, preferably, a VL region comprising the sequence of SEQ ID NO: 152 (017)
i) a VH region comprising the sequence of SEQ ID NO: 153 and, preferably, a VL region comprising the sequence of SEQ ID NO: 154 (025), or
j) a variant of any one of said antibodies, said variant preferably having at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.
[0081] In another main embodiment of the antibody of the invention:
- the antibody competes to bind soluble cMetECDHis with an immobilized antibody, said immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 49 and preferably a VL region comprising the sequence of SEQ ID NO: 53 (045) the antibody competing in more than 50%, such as more than 75% with said immobilized antibody, when determined as described in Example 17, and
- the antibody does not compete to bind soluble cMetECDHis with an immobilized antibody, said immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 13 (006), preferably the antibody competing in less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
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33/121 [0082] In yet another embodiment, the antibody does not compete to bind soluble cMetECDHis with an antibody selected from the group consisting of:
a) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 21 (008), and
b) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 37 (024), preferably the antibody competing in less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
[0083] In yet another embodiment, the antibody binds to the same epitope as that of an antibody comprising a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 53 (045) .
[0084] In yet another embodiment, the antibody comprises a VH CDR3 region having the sequence as shown in SEQ ID NO: 188, as well as a VH CDR3 region as shown in SEQ ID NO: 52 (045).
[0085] In yet another embodiment, the antibody comprises a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 186, 187 and 188 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO : 54, 55 and 56, such as an antibody comprising a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 50, 51 and 52 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 54, 55 and 56 (045).
[0086] In yet another embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO: 49 and, preferably, a VL region comprising the sequence of
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SEQ ID NO: 53 (045)
b) a VH region comprising the sequence of SEQ ID NO: 155 and, preferably, a VL region comprising the sequence of SEQ ID NO: 156 (040)
c) a VH region comprising the sequence of SEQ ID NO: 157 and, preferably, a VL region comprising the sequence of SEQ ID NO: 158 (039), or
d) a variant of any one of said antibodies, said variant preferably having at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.
[0087] In yet another embodiment, the antibody binds in the SEMA domain of c-Met, preferably the antibody being able to inhibit HGF binding in the SEMA domain with an IC 50 of less than 10 pg / ml, such as less than 2 pg / ml as described in Example 9.
[0088] In another main embodiment of the antibody of the invention, the antibody binds in the same epitope as that of an antibody comprising a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 21 (008) or binds in the same epitope as that of an antibody comprising a VH region comprising the sequence of SEQ ID NO: 41 and a VL region comprising the sequence of SEQ ID NO: 45 (035) or binds in same epitope as that of an antibody comprising a VH region comprising the sequence of SEQ ID NO: 105 and a VL region comprising the sequence of SEQ ID NO: 109 (096).
[0089] In yet another embodiment, the antibody comprises a VH CDR3 region having the sequence as shown in SEQ ID NO: 183, such as a VH CDR3 region as shown in SEQ ID NO: 20, 44 or 108 (008, 035, 096).
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35/121 [0090] In yet another embodiment, the antibody comprises a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 18, 182 and 183 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 22, 203 and 204, such as an antibody comprising
a) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 18, 19 and 20 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 22, 23 and 24, (008 ), or
b) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 42, 43 and 44 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 46, 47 and 48, (035 ), or
c) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 106, 107 and 108 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 110, 111 and 112 (096) .
[0091] In yet another embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO: 17 and, preferably, a VL region comprising the sequence of SEQ ID NO: 21 (008)
b) a VH region comprising the sequence of SEQ ID NO: 41 and, preferably, a VL region comprising the sequence of SEQ ID NO: 45 (035)
c) a VH region comprising the sequence of SEQ ID NO: 105 and, preferably, a VL region comprising the sequence of SEQ ID NO: 109 (096) or
d) a variant of any of said antibodies, said variant preferably having at most 1 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.
[0092] In yet another embodiment, the antibody binds to A431 cells with an EC50 of 10 nM or less, such as an EC50 of 2 nM or
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36/121 less, preferably as determined according to Example 13.
[0093] In yet another embodiment, the antibody binds to c-Met with an affinity constant (Kd) of 20 nM or less, such as an affinity of 5 nM or less, preferably as determined according to Example 14 .
[0094] In yet another embodiment, the antibody binds to Rhesus c-Met, preferably the antibody binding signal to Rhesus cMet is at least 5 times that of a negative control antibody, as determined by according to Example 15.
[0095] In yet another embodiment, the antibody inhibits the binding of HGF in the extracellular domain of c-Met, preferably with the antibody inhibiting binding by more than 40%, such as more than 50%, e.g. more than 60%, e.g. more than 70%, e.g. more than 80%, e.g. more than 90%, as determined according to Example 16.
[0096] In yet an additional embodiment, the antibody is able to inhibit the viability of KP4 cells, preferably with the antibody being able to inhibit viability by more than 10%, such as more than 25%, e.g. more than 40%, preferably as described in Example 19.
Antibody formats [0097] The present invention provides antagonistic and non-antagonistic anti-c-Met antibodies. Although some antibodies act antagonistically on target cells regardless of whether they are monovalent or divalent, for other antibodies, the functional effect depends on the valence. As shown here in Example 19, antibodies 024, 062, 064, 068, 069, 098, 101, 181, for example, (which are all within the same cross-block group see Example 17) have antagonistic properties in a viability assay of KP4 regardless of format. Antibodies 022 and 058, on the other hand, behave antagonistically in this assay in a monovalent format, but agonistically (or at least not antagonistically)
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37/121 in a bivalent format. Thus, depending on the functional properties for a special use, special antibodies can be selected from the set of antibodies provided in the present invention and / or their format can be adapted to change the valence.
[0098] Furthermore, the antibody of the invention can be of any isotype. The choice of isotype will typically be driven by the desired effector functions, such as induction of ADCC. Exemplary isotopes are IgG1, IgG2, IgG3, and IgG4. Any human, kappa or lambda light chain constant regions can be used. If desired, the class of an anti-c-Met antibody of the present invention can be switched by known methods. For example, an antibody of the present invention that was originally IgM may have the class switched to an IgG antibody of the present invention. In addition, class switching techniques can be used to convert an IgG subclass into another, for example from IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention can be modified by switching from isotype to, e.g. , an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment an antibody of the present invention is an IgG1 antibody, for example an IgG1, K.
[0099] C-Met inframodulation induced by antagonistic antibodies represents a mechanism of action of therapeutic anti-c-Met antibodies. Consequently, in one aspect of the invention antibodies with reduced agonistic properties, but with a retained ability to induce c-Met inframodulation are desirable.
[00100] It has been revealed that by reducing the conformational flexibility of the antibodies the potential residual agonistic activities of the divalent IgG1 antibodies are minimized.
[00101] Consequently, in yet another embodiment, the antibody of the invention has been modified to become more flexible, such as
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38/121 mutations in the articulation region.
[00102] Major conformational changes are the result of joint flexibility, which allows a wide variety of Fab-Fc angles (Ollmann Saphire, E., RL Stanfield, MDM Crispin, PWHI Parren, PM Rudd, RA Dwek, DR Burton and IA Wilson, 2002. “Contrasting IgG structures reveal extreme asymmetry and flexibility.” J. Mol. Biol. 319: 9-18). One way to reduce the flexibility of the Fab arm in immunoglobulins is to prevent the formation of disulfide bonds between the light chain and the heavy chain through genetic modification. In a natural IgG1 antibody the light chain is covalently connected to the heavy chain via a disulfide bond, connecting the C-terminal cysteine of the light chain to the cysteine at position 220 (EU number C220) at the Fc joint of the heavy chain. Whether by mutating the amino acid C220 to serine or another natural amino acid, or by removing C220, removing the entire joint, or replacing the IgG1 joint with an IgG3 joint, a molecule is formed in which the light chains are connected via its C-terminal cysteines, analogous to the situation found in the human IgA2m isotype (1). This results in a reduced flexibility of the Fabs in relation to the Fc and consequently in reduced crosslinking capacity, as shown in the Examples.
[00103] Another strategy to reduce the flexibility of an IgG1 molecule is to replace the IgG1 joint with the IgG2 joint or with the IgG2 similar joint. (Dangl et al. EMBO J. 1988; 7: 1989-94). This region of articulation has two distinct properties from that of IgG1, which are considered to make molecules less flexible. First, compared to the IgG1 joint, the IgG2 joint is 3 amino acids shorter. Second, the IgG2 joint contains an additional cysteine, so three will be formed instead of two heavy interchain disulfide bridges. Alternatively, a variant of the IgG1 joint that looks like
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39/121 with the IgG2 joint can be introduced. This mutant (ΤΗ7Δ6-9) (WO2010063746) contains a T223C mutation and two deletions (K222 and T225) in order to create a shorter joint with an additional cysteine.
[00104] In yet another embodiment, the antibody of the invention is of the IgG1 subtype, in which the joint has been modified by:
(i) deletion of the region of articulation of the EPKSCDKTHTCPPCP sequence and its replacement by the region of articulation of IgG2 of the sequence: ERKCCVECPPCP (IgG1-IgG2);
(ii) deletion of position 220 so that the joint region has the sequence of EPKSDKTHTCPPCP (IgG1 ΔC220);
(iii) replacement of cysteine at position 220 by any other natural amino acid (X) so that the articulation region has the sequence of EPKSXDKTHTCPPCP (IgG1 C220X);
(iv) deletion of the sequence joint region
EPKSCDKTHTCPPCP (UniBody IgG1);
(v) deletion of the region of articulation of the sequence
EPKSCDKTHTCPPCP and its replacement by the IgG3 joint region of the sequence
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK SCDTPPPCPRCP (IgG1 joint-IgG3 '); or (vi) replacement of threonine at position 223 with cysteine, and deletion of lysine at position 222 and threonine at position 225, so that the joint region has the sequence of EPKSCDCHCPPCP (IgG1 ΤΗ7Δ6-9).
[00105] In one embodiment of the invention, the antibody of the invention is of the IgG1 subtype, in which the hinge region has been modified by deleting position 220 so that the hinge region has the sequence of EPKSDKTHTCPPCP (IgG1 ΔC220) or by replacement of cysteine in
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40/121 position 220 by any other natural amino acid (X) so that the articulation region has the sequence of EPKSXDKTHTCPPCP (IgG1 C220X);
[00106] In yet another embodiment, the antibody of the invention is of the IgG1 subtype, in which the hinge region has been modified by replacing the cysteine in position 220 by serine so that the hinge region has the sequence of EPKSSDKTHTCPPCP (IgG1 C220S ). In yet another embodiment, the antibody of the invention is of IgG2 subtype.
[00107] In yet another embodiment, the antibody of the invention is glycoengineered to reduce fucose and thereby intensify ADCC, e.g. by adding compounds to the culture medium during antibody production as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105: 350 or by using inactivated FUT8 cells, p. eg, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87: 614. ADCC can alternatively be optimized using the method described by Umana et al. (1999) Nature Biotech. 17: 176.
[00108] In one embodiment, the antibody comprises a VH region comprising the CDR3 sequence of SEQ ID NO: 100 and a VL region comprising the CDR3 sequence of SEQ ID NO: 104 (069) of the IgG1 subtype, in which the region of articulation has been modified by substituting the cysteine in position 220 for serine so that the articulation region has the sequence of EPKSSDKTHTCPPCP (IgG1 C220S).
[00109] In one embodiment, the antibody comprises a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 98, 99 and 100 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 102, 103 and 104 (069) of the IgG1 subtype, in which the articulation region has been modified by substituting the cysteine in position 220 for serine so that the articulation region has the sequence of EPKSSDKTHTCPPCP (IgG1 C220S).
[00110] In one embodiment, the antibody comprises a VH region
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41/121 comprising the sequence of SEQ ID NO: 97 and a VL region comprising the sequence of SEQ ID NO: 101 (069) of the IgG1 subtype, in which the hinge region has been modified by substituting the cysteine in position 220 for serine so that the hinge region has the sequence of EPKSSDKTHTCPPCP (IgG1 C220S).
[00111] Several publications have demonstrated the correlation between nucleus fucosylation and enhanced ADCC activity in vitro (Shields RL. 2002 JBC; 277: 26733-26740, Shinkawa T. 2003 JBC; 278 (5): 3466-3473, Umana P Nat. Biotechnol. 1999 Feb; 17 (2): 176-80).
[00112] In yet another embodiment, the antibody of the invention has been modified to reduce core fucosylation below 10%, such as below 5% as determined with high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAECPAD) . This can be accomplished by methods well known in the prior art, e.g. eg, treatment with kifunensin or production in FUT8 negative cells.
[00113] In yet another embodiment, the antibody of the invention has been engineered to enhance complement activation, e.g. , as described in Natsume et al. (2009) Cancer Sci. 100: 2411.
[00114] In one embodiment, the antibody of the invention is a full length antibody, preferably an IgG1 antibody, in particular an IgG1, K antibody. In another embodiment, the antibody of the invention is an antibody fragment or a single chain antibody.
[00115] Antibody fragments can be e.g. , obtained by fragmentation using conventional techniques, and fragments selected for utility in the same manner as described herein for whole antibodies. For example, F (ab'b fragments can be generated by treating the antibody with pepsin. The resulting F (ab ') 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Fab fragments can be obtained by treatment of an IgG antibody with
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42/121 papain; Fab 'fragments can be obtained with pepsin digestion of the IgG antibody. An F (ab ') fragment can also be produced by Fab' bond described below via a thio-ether bond or a disulfide bond. A Fab 'fragment is an antibody fragment obtained by cutting a disulfide bond from the F (ab') 2 hinge region. A Fab 'fragment can be obtained by treating an F (ab') 2 fragment with a reducing agent, such as dithiothreitol. Antibody fragment can also be generated by the expression of nucleic acids encoding such fragments in recombinant cells (see for example Evans et al., J. Immunol. Meth. 184. 123-38 (1995)). For example, a chimeric gene encoding a portion of the F (ab ') 2 fragment could include DNA sequences encoding the CH1 domain and the H chain articulation region, followed by a translation termination codon to give such a molecule of truncated antibody fragment.
[00116] As explained above, in one embodiment, the anti-Met antibody of the invention is a bivalent antibody.
[00117] In another embodiment, the anti-c-Met antibody of the invention is a monovalent antibody.
[00118] In one embodiment, the antibody of the invention is a Fab fragment or an arm antibody, as described in US20080063641 (Genentech) or other monovalent antibody, e.g. , as described in WO2007048037 (Amgen).
[00119] In a preferred embodiment, a monovalent antibody has a structure as described in WO2007059782 (Genmab) (incorporated herein by reference) having a deletion of the hinge region. Consequently, in one embodiment, the antibody is a monovalent antibody, said anti-c-Met antibody being constructed by a method comprising:
i) obtain a nucleic acid construct encoding the strand
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43/121 light of said monovalent antibody, said construct comprising a nucleic acid sequence encoding the VL region of an antibody specific for selected antigen anti-c-Met and a nucleotide sequence encoding the Ig region constant CL, said nucleotide sequence encoding the VL region of an antibody specific for the selected antigen and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and in the case of an IgG1 subtype, the nucleotide sequence encoding the region CL has been modified in such a way that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising a sequence of identical amino acids from the CL region in the presence of human polyclonal IgG or when administered to an animal or human being;
ii) obtain a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of an antibody specific for the selected antigen and a nucleotide sequence encoding a CH region constant of an Ig of human, and the nucleotide sequence encoding the CH region has been modified in such a way that the region corresponding to the articulation region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, do not comprise any residues of amino acids that participate in the formation of disulfide bonds or stable covalent or non-covalent bonds of heavy chains with other peptides comprising an identical amino acid sequence of the CH region of human Ig in the presence of polyclonal human IgG or when administered to an animal or being human being, that said sequence of nucleus tides encoding the VH region of an antibody specific for the selected antigen and said nucleotide sequence encoding the CH region of said Ig are operatively
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44/121 connected together;
iii) obtaining a cell expression system to produce said monovalent antibody;
iv) producing said monovalent antibody by coexpressing the (i) and (ii) nucleic acid constructs in cells of the (iii) cell expression system.
Similarly, in one embodiment, the anti-c-Met antibody is a monovalent antibody, comprising (i) a variable region of an antibody of the invention as described herein or an antigen binding part of said region, and ( ii) a CH region of an immunoglobulin or a fragment thereof comprising the Ch2 and Ch3 regions, the Ch region or its fragment having been modified in such a way that the region corresponding to the articulation region and, if the immunoglobulin is not a subtype IgG4, other regions of the CH region, such as the CH3 region, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH region or other stable covalent or non-covalent heavy interchanges with an identical CH region in the presence of human polyclonal IgG.
[00121] In yet another modality, the heavy chain of the monovalent anti-c-Met antibody has been modified in such a way that the entire joint has been deleted.
[00122] In another additional embodiment, said monoclonal antibody is of the IgG4 subtype, but the CH3 region has been modified so that one or more of the following amino acid substitutions have been made:
_____________________ Numbering of CH3 mutations
KABAT * EU G4 Index * Mutations
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E378 E357 E357A or E357T or E357V or E357I S387 S364 S364R or S364K T389 T366 T366A or T366R or T366K or T366N L391 L368 L368A or L368V or L368E or L368G orL368S or L368T D427 D399 D399A or D399T or D399S F405A orF405L or F405T or F405D or F405R orF405Q F436 F405 or F405K or F405Y Y438 Y407 Y407A or Y407E or Y407Q or Y407K orY407F F436 and Y438 F405 and Y407 (F405T and Y407E) or (F405D and Y407E) D427 and Y438 D399 and Y407 (D399S and Y407Q) or (D399S and Y407K) or
(D399S and Y407E) * KABAT indicates amino acid numbering according to Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest”, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) EU index indicates amino acid numbering according to EU index as summarized in Kabat et al., “Sequences of Proteins of Immunological Interest”, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) .
In another additional embodiment, the sequence of said monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked glycosylation.
[00124] Anti-c-Met antibodies of the invention also include single chain antibodies. Single chain antibodies are peptides to which the heavy and light chain Fv regions are connected. In one embodiment, the present invention provides a single chain Fv (Fv, scFv) with the heavy and light chains on the Fv of an anti-c-Met antibody of the present invention being joined with a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of producing such antibodies are described in, for example, US 4,946,778, Pluckthun in "The Pharmacology of Monclonal Antibodies", vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al.,
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PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature 348. 552-554 (1990). The single chain antibody can be monovalent, if only a single Vh and Vl are used, bivalent, if two Vh and Vl are used, or polyvalent, if more than two VH and VL are used.
[00125] In one embodiment, the anti-c-Met antibody of the invention is an antibody deficient in effector function. In one embodiment, the effector function deficient antic-Met antibody is a stabilized IgG4 antibody, which has been modified to prevent Fab arm replacement (van der Neut Kolfschoten et al. (2007) Science 317 (5844): 1554-7 ). Examples of suitable stabilized IgG4 antibodies are antibodies, in which arginine at position 409 in a human IgG4 heavy chain constant region, which is indicated in the EU index as in Kabat et al., Is replaced by lysine, threonine, methionine, or leucine, preferably lysine (described in WO2006033386 (Kirin)) and / or in which the hinge region has been modified to comprise a Cys-Pro-Pro-Cys sequence.
[00126] In yet another embodiment, the stabilized anti-c-Met IgG4 antibody is an IgG4 antibody comprising a heavy chain and a light chain, in which said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of of: Lys, Ala, Thr, Met and Leu in the position corresponding to 409 and / or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu in the position corresponding to 405, and that said antibody optionally comprises a or more substitutions, deletions and / or additional insertions, but does not comprise a Cys-Pro-Pro-Cys sequence in the joint region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2 or human IgG3. See also WO2008145142 (Genmab).
[00127] In yet another embodiment, the IgG4 anti-c-Met antibody
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Stabilized 47/121 is an IgG4 antibody comprising a heavy chain and a light chain, in which said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of: Lys, Ala, Thr, Met and Leu in position corresponding to 409 and / or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu in the position corresponding to 405, and said antibody optionally comprising one or more additional substitutions, deletions and / or insertions and being that said antibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, human IgG2 or human IgG3.
[00128] In yet another embodiment, the effector function deficient anti-c-Met antibody is an antibody of a non-IgG4 type, p. eg, IgG1, IgG2 or IgG3 that has been mutated in such a way that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have been described, e.g. in Dall'Acqua WF et al., J. Immunol. 177 (2): 1129-1138 (2006) and Hezareh M, J. Virol .; 75 (24): 12161-12168 (2001).
Conjugates [00129] In yet another embodiment, the present invention provides an anti-c-Met antibody conjugated to a therapeutic group, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressive agent, or a radioisotope. Such conjugates are here called "immunoconjugates". Immunoconjugates that include one or more cytotoxins are called "immunotoxins".
[00130] A cytotoxin or cytotoxic agent includes any agent that is harmful (eg, exterminates) cells. Suitable therapeutic agents to form immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin,
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48/121 etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracine-dione, mitoxantrone, mitramicine, actinomycin D, 1-dehydro-testosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, propranolol, propranolol , antimetabolites (such as methotrexate, 6-mercapto-purine, 6-thio-guanine, cytarabine, fludarabine, 5-fluoro-uracil, decarbazine, hydroxy-urea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechatramine , chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as antibiotics (such as carbohydrates) (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (such as diphtheria A chain and its active fragments and hybrid molecules), ricin toxin (such as ricin A or deglycosylated ricin A toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II , SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soy Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorine, saporin, modeccin, gelanin, abrina A chain, chain Modeccin, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, American Phytolacca proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcine, crotin, Saponaria officinalis inhibitor, gelonin toxins, mitogelin, restrictocin, phenomycin, and enomycin. Other suitable conjugated molecules include ribonuclease (RNase), DNase I, enterotoxin-A staphylococal, antiviral protein from cancers, diterin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents, which can be administered in combination with an anti-c-Met antibody from
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49/121 present invention as described herein elsewhere, may also be candidates for therapeutic groups useful for conjugation to an antibody of the present invention.
[00131] In another embodiment, an anti-c-Met antibody of the invention comprises a conjugated nucleic acid or a molecule associated with a conjugated nucleic acid. In such a facet of the present invention, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (eg, a siRNA molecule) or an immunostimulating nucleic acid (eg, an DNA molecule containing the immunostimulating CpG motif). In another embodiment, an anti-c-Met antibody of the invention is conjugated to an aptamer or a ribozyme.
[00132] In one embodiment, anti-c-Met antibodies are obtained comprising one or more radiolabeled amino acids. A radiolabeled anti-Met antibody can be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of markers for polypeptides include 3h 14c 15n 35S 90y 99m „_ 125t 131t„ I86q „,,,,, c, e,, and e.
[00133] Anti-c-Met antibodies can also be chemically modified by covalent conjugation to a polymer for example to increase their circulation half-life. Exemplary polymers, and methods for linking them into peptides, are illustrated for example in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional polymers include polyoxyethylated polyols and poly (ethylene glycol) (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).
Any method known in the art to conjugate the anti-c-Met antibody to conjugated molecule (s), such as those described above, can be used, including the methods described by Hunter et al., Nature 144. 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J.
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Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such antibodies can be produced by chemical conjugation of another group on the N-terminal side or on the C-terminal side of the anti-c-Met antibody or its fragment (eg, an H or L chain of anti-c-Met antibody ) (see, eg, “Antibody Engineering Handbook”, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated antibody derivatives can also be generated in sugars or internal residues, if appropriate. The agents can be copulated either directly or indirectly into an anti-c-Met antibody of the present invention. An example of indirect copulation of a second agent is copulation by a spacer group. In one embodiment, the anti-c-Met antibody of the present invention is linked to a chelating linker, e.g. eg, tiuxetan, which allows the antibody to be conjugated to a radioisotope.
Bispecific antibodies [00135] In a further aspect, the invention relates to a bispecific molecule comprising a first antigen binding site of an anti-c-Met antibody of the invention as described hereinabove and a second antigen binding site with a different binding specificity, such as a binding specificity for a human effector cell, a human Fc receptor, a T cell receptor, a B cell receptor, or a binding specificity for a non-overlapping epitope of c-Met , ie a bispecific antibody in which the first and second antigen binding sites do not compete for c-Met binding, e.g. when tested as described in Example 17.
Exemplary bispecific antibody molecules of the invention comprise (i) two antibodies, which are conjugated together, one with a specificity for c-Met and another for a second target, (ii) a single antibody having a chain or an arm
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51/121 specific (o) for c-Met and a second chain or a second specific arm (o) for a second molecule, and (iii) a single chain antibody that has specificity for c-Met and a second molecule. In one embodiment, the second molecule is a tumor-associated antigen / cancer antigen such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), α-fetoprotein, CAMEL (antigen recognized by CTL on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (eg, MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7 , Ep-CAM or a cancer-associated integrin, such as α5β3 integrin. In another embodiment, the second molecule is an angiogenic factor or another cancer growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin or a receptor for any of these, especially receptors associated with cancer progression (for example one of the HER1-HER4 receptors). In one embodiment, a bispecific antibody of the present invention is a diabody.
Nucleic acid sequences, vectors and host cells [00137] In a further aspect, the invention relates to nucleic acid sequences, such as DNA sequences, encoding heavy and light chains of an antibody of the invention.
[00138] In one embodiment, the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45 , 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 138, 139 , 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 , 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178.
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52/121 [00139] In another special embodiment, the nucleic acid sequence encodes a VH amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65 , 73, 81, 89, 97, 105, 113, 121, 129, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,
159, 161, 163, 165, 167, 169, 171, 173, 175 and 177.
[00140] In another special embodiment, the nucleic acid sequence encodes a VL amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 164, 166, 168, 170, 172, 174, 176 and 178.
[00141] In a further aspect, the invention relates to an expression vector, or a set of expression vectors, encoding an antibody of the invention. The heavy and light chains of the antibody can be encoded by the same or different vector.
[00142] Such expression vectors can be used for recombinant production of antibodies of the invention.
[00143] In one embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1, 5, 9, 13, 17, 21, 25 , 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125 , 129, 133, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 , 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178.
[00144] In another special embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VH amino acid sequences selected from the group consisting of: SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
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53/121
165, 167, 169, 171, 173, 175 and 177.
[00145] In another special embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VL amino acid sequences selected from the group consisting of: SEQ ID NO: 5, 13, 21, 29, 37, 45 , 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 , 166, 168, 170, 172, 174, 176 and 178.
[00146] In yet another embodiment, the expression vector additionally comprises a nucleotide sequence encoding the constant region of a light chain, a heavy chain, or both light and heavy chains of an antibody, e.g. , a human antibody.
[00147] An expression vector in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, an anti-c-Met antibody-encoding nucleic acid is comprised of a naked DNA or RNA vector, including, for example, a linear expression element (as described in for example Sykes and Johnston, Nat. Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (as described in for example US 6,077,835 and / or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC118 / 119, a minimally "midge" nucleic acid vector (as described in for example Schakowski et al., Mol. Ther. 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a construct precipitated by CaPO4 ^- (as described in for example WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler
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54/121 et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see for example US 5,589,466 and US 5,973,972).
[00148] In one embodiment, the vector is suitable for expression of the anti-c-Met antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN (Van Heeke & Schuster, J. Biol. Chem. 264. 5503-5509 (1989), pET (Novagen, Madison WI) and the like) vectors.
[00149] An expression vector can also or alternatively be a suitable vector for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., Ed. “Current Protocols in Molecular Biology”, Greene Publishing and Wiley InterScience Nova York (1987), and Grant et al., Methods in Enzymol. 153, 516-544 (1987)).
[00150] An expression vector can also or alternatively be a suitable vector for expression in mammalian cells, e.g. a vector comprising glutamine synthase as a selectable marker, such as the vectors described in (Bebbington (1992) Biotechnology (NY) 10: 169-175).
[00151] A nucleic acid and / or vector may also comprise a nucleic acid sequence encoding a secretion / location sequence, which may select a polypeptide, such as a nascent polypeptide chain, in the periplasmic space or into the media. cell culture. Such sequences are known in the art, and include signal peptides or expression leaders.
[00152] In an expression vector of the invention, nucleic acids encoding anti-c-Met antibody may comprise or be associated with any promoter, enhancer or other elements
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55/121 suitable facilitators of expression. Examples of such elements include strong promoters (eg, human CME IE enhancer / promoter as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (eg, a polylinker). Nucleic acids can also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.
[00153] In one embodiment, the expression vector encoding anti-c-Met antibody may be positioned in and / or released to the host cell or host animal via a viral vector.
[00154] In a further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of the invention as defined herein. Examples of host cells include yeast, bacterial, and mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the present invention provides a cell comprising a nucleic acid stably integrated into the cell genome that comprises a coding sequence for expression of an anti-c-Met antibody of the present invention. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence encoding an anti-c-Met antibody of the invention.
[00155] In a further aspect, the invention relates to a hybridoma that produces an antibody of the invention as defined herein. In a further aspect, the invention relates to a transgenic non-human animal or a transgenic plant comprising nucleic acids encoding a human heavy chain and a human light chain, the animal or plant producing an antibody from invention of the invention.
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56/121 [00156] In a further aspect, the invention relates to a method for producing an anti-c-Met antibody of the invention, said method comprising the steps of
a) cultivating a hybridoma or host cell of the invention as described above, and
b) purifying the antibody of the invention from the culture medium.
Compositions [00157] In a further main aspect, the invention relates to a pharmaceutical composition comprising:
- an anti-c-Met antibody as defined herein, and
- a pharmaceutically acceptable vehicle.
[00158] The pharmaceutical composition of the present invention may contain an antibody of the present invention or a combination of different antibodies of the present invention.
[00159] The pharmaceutical compositions may be formulated according to conventional techniques such as those disclosed in "Remington: The Science and Practice of Pharmacy", 19 ^th Edition, Gennaro, Ed, Mack Publishing Co., Easton, Pa . , 1995. A pharmaceutical composition of the present invention can e.g. include diluents, fillers, salts, buffers, detergents (eg, a non-ionic detergent, such as Tween-20 or Tween80), stabilizers (eg, sugars or protein-free amino acids), preservatives, tissue fixatives, solubilization, and / or other materials suitable for inclusion in a pharmaceutical composition.
[00160] Pharmaceutically acceptable vehicles include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption retardants, and the like that are physiologically compatible with a compound of the present invention. Examples of suitable aqueous and non-aqueous vehicles that can be used in pharmaceutical compositions
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57/121 of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, poly (ethylene glycol), and the like), and suitable mixtures thereof, oils vegetables, colloidal solutions of carboxymethylcellulose, tragacanth gum and injectable organic esters, such as ethyl oleate, and / or various buffers. Pharmaceutically acceptable vehicles include sterile and post-sterile aqueous dispersions or solutions for the extemporaneous preparation of dispersion or sterile injectable solutions. Proper fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.
[00161] Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for example (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxy-anisol, butylated hydroxy-toluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylene-diamino-tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[00162] Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
[00163] The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which can enhance half-life or effectiveness pharmaceutical composition. The compounds of the present invention can be prepared with vehicles that will protect the compound against rapid release, such as a formulation of
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58/121 controlled release, including implants, transdermal patches, and microencapsulated delivery systems. Such vehicles may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene-vinyl acetate polymers, polyanhydrides, poly (glycolic acid), collagen, poly-polyesters, and poly (lactic acid) alone or with a wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art.
[00164] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with an ingredient or a combination of ingredients p. eg as listed above, if required, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other required ingredients e.g. from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization) which gives a powder of the active ingredient plus any desired additional ingredient from a previously sterile filtered solution.
[00165] The actual dosage levels of the active ingredients in pharmaceutical compositions can be varied in order to obtain an amount of the active ingredient that is effective in achieving the therapeutic response for a special patient, special composition, and special mode of administration, without be toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the special compositions of the present invention used, or their amide, the route of administration, the rate of excretion of the special compound being used, the duration of treatment, others ( the)
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59/121 drugs, compounds and / or materials used in combination with the special compositions used, age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in medical arts.
[00166] The pharmaceutical composition can be administered by any suitable route or mode (o). In one embodiment, a pharmaceutical composition of the present invention is administered parenterally. "Administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratrendeal, transtraqueal, subcutaneous injection , subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal and infusion.
[00167] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or by infusion.
Uses [00168] In yet another main aspect, the invention relates to an anti-c-Met antibody of the invention for use as a medicament.
[00169] The anti-c-Met antibodies of the invention can be used for numerous purposes. In particular, the antibodies of the invention can be used to treat various forms of cancer, including metastatic cancer and refractory cancer. Such cancer may be dependent on HGF or independent of HGF.
[00170] In one embodiment, the anti-c-Met antibodies of the invention are used to treat a form of cancer selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer , esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, cancer
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60/121 lung (such as non-small cell lung cancer (NSCLC)), nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer and thyroid cancer.
[00171] In another embodiment, the anti-c-Met antibodies of the invention are used to treat a form of cancer selected from the group consisting of: osteosarcoma, rhabdomyosarcoma and synovial sarcoma.
[00172] In another embodiment, the anti-c-Met antibodies of the invention are used to treat a form of cancer selected from the group consisting of: Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma and fibrosarcoma.
[00173] In another embodiment, the anti-c-Met antibodies of the invention are used for the treatment of hematopoietic malignancies, such as a malignancy selected from the group consisting of: acute myelogenous leukemia, adult T-cell leukemia, chronic myeloid leukemia, lymphoma and multiple myeloma.
[00174] In yet another embodiment, the anti-c-Met antibodies of the invention are used for the treatment of a neoplasm selected from the group consisting of: glioblastoma, astrocytoma, melanoma, mesothelioma and Wilm's tumor.
[00175] In yet another embodiment, the anti-c-Met antibodies of the invention are used for the treatment of MiT tumors, including clear cell sarcoma (CCS), soft tissue alveolar sarcoma (ASPS) and renal cell sarcoma associated with translocation.
[00176] In another embodiment, agonistic anti-c-Met antibodies of the invention are used for the regulation of cytokine production and the induction of endothelial progenitor cell mobilization, e.g. in patients with coronary heart disease (Yang et al. (2009) Clin. Exp. Pharmacol. Physiol. 36: 790).
[00177] In another embodiment, agonistic anti-c-Met antibodies from
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61/121 invention are used to inhibit or ameliorate chronic kidney failure (Mizuno et al. (2008) Front Biosci. 13: 7072).
[00178] Similarly, the invention relates to a method for inhibiting the growth and / or proliferation of a tumor cell expressing c-Met, comprising administering to an individual in need thereof an effective amount of an antibody of the invention.
[00179] In one embodiment, said tumor cell is involved in a form of cancer selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, cancer head and neck cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, Kaposi's sarcoma, leiomyosarcoma , malignant fibrous histiocytoma, fibrosarcoma, acute myelogenous leukemia, adult T-cell leukemia, chronic myeloid leukemia, lymphoma, multiple myeloma, glioblastoma, astrocytoma, melanoma, mesothelioma and Wilm's tumor.
[00180] Also, the invention relates to the use of a monoclonal antibody that binds to human c-Met for the preparation of a medicament for the treatment of cancer, such as one of the specific cancer indications mentioned above.
[00181] In one embodiment, selection of patients to be treated with an anti-c-Met antibody is based on the level of (supra) expression of cMet and / or HGF on the relevant tumor cells of said patients.
[00182] In yet another embodiment of the treatment methods of the present invention, treatment effectiveness is being monitored during therapy, e.g. e.g., at predetermined times of time, by determining levels of c-Met expression on the relevant tumor cells.
[00183] Dosage regimens in the treatment methods and uses above
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62/121 are adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single cake can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. Parenteral compositions can be formulated in unit dosage form to facilitate administration and uniformity of dosage.
[00184] Efficient dosages and dosage regimens for anti-c-Met antibodies depend on the disease or condition to be treated and can be determined by persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1 -20 mg / kg, such as about 0.1-10 mg / kg, for example about 0.5, about such as 0.3, about 1, about 3, about 5, or about 8 mg / kg.
[00185] A doctor or veterinarian having common experience in the art can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the doctor or veterinarian could start with doses of the anti-c-Met antibody used in the pharmaceutical composition at levels lower than those required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present invention will be that amount of the compound which is the lowest effective dose to produce a therapeutic effect. Administration can p. be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, anti-c-Met antibodies can be administered by infusion at a weekly dosage of 10 to 500 mg / m ² , such as from 200 to 400 mg / m ² . Such administration can be repeated, e.g. 1 to 8 times, such as 3 to 5 times. Administration can be carried out by continuous infusion over a period of
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63/121 period of 2 to 24 hours, such as 2 to 12 hours. In one embodiment, anti-c-Met antibodies can be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.
[00186] In one embodiment, anti-c-Met antibodies can be administered in a weekly dosage of 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for example up to 8 times, such as 4 to 6 times. Such a regimen can be repeated one or more times if necessary, for example, after 6 months or 12 months. The dosage can be determined or adjusted by measuring the amount of the compound of the present invention in the blood under administration for example by collecting a biological sample and using anti-idiotypic antibodies that select the antigen-binding region of the anti-c-Met antibodies of the present invention.
[00187] In one embodiment, anti-c-Met antibodies can be administered by maintenance therapy, such as, e.g. eg, once a week for a period of 6 months or more.
[00188] An anti-c-Met antibody can also be administered prophylactically for the purpose of reducing the risk of developing cancer, delaying the onset of the occurrence of a cancer progression event, and / or reducing the risk of recurrence when a cancer is in remission.
[00189] Anti-c-Met antibodies can also be administered in combination therapy, i.e., combined with other therapeutic agents relevant to the disease or condition being treated. Consequently, in one embodiment, the antibody-containing drug is for combination with one or more additional therapeutic agents, such as a cytotoxic, chemotherapeutic or antiangiogenic agent.
[00190] Such combined administration can be simultaneous, separate or sequential. For simultaneous administration the agents can be administered as a composition or as separate compositions, if
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64/121 appropriate. The present invention therefore provides methods for treating a disorder involving cells expressing c-Met as described above, whose methods comprise administration of an anti-c-Met antibody of the present invention combined with one or more additional therapeutic agents as described below.
[00191] In one embodiment, the present invention provides a method for treating a disorder involving cells expressing c-Met in a subject, the method of which comprises administering a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least an additional therapeutic agent to a subject in need of it.
[00192] In one embodiment, the present invention provides a method for treating or preventing cancer, the method of which comprises administering a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least one additional therapeutic agent to a subject in need for it.
[00193] In one embodiment, such an additional therapeutic agent can be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thio-guanine, cytarabine, fludarabine, 5-fluoro-uracil, decarbazine, hydroxy-urea, asparaginase , gemcitabine or cladribine.
[00194] In another embodiment, such an additional therapeutic agent can be selected from an alkylating agent, such as meclorethamine, thiope, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromo-mannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin.
[00195] In another embodiment, such an additional therapeutic agent can be selected from an antimitotic agent, such as taxanes, for example docetaxel, and paclitaxel, and vinca alkaloids, for example vindesine, vincristine, vinblastine, and vinorelbine.
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65/121 [00196] In another embodiment, such an additional therapeutic agent can be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.
[00197] In another embodiment, such an additional therapeutic agent can be selected from a growth factor inhibitor, such as an ErbB1 (EGFR) inhibitor (such as an anti-EGFR antibody, e.g., zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), an ErbB2 inhibitor (Her2 / neu) (such as an anti-HER2 antibody, eg, trastuzumab, trastuzumab-DM1 or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).
[00198] In another embodiment, such an additional therapeutic agent can be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib, PTK787 / ZK222584.
[00199] In another embodiment, the present invention provides a method for treating a disorder involving cells expressing c-Met in a subject, the method of which comprises administering a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least an inhibitor of angiogenesis, neovascularization, and / or other vascularization in a subject in need of it.
[00200] Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP470, squalamine, 2-methoxy-estradiol, combretastatin, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plow Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and similar agents) , antagonists of angiogenic growth factors (such as ZD6474, SU6668, antibodies against angiogenic agents and / or their receptors (such as VEGF (eg, bevacizumab), bFGF, and angiopoietin-1),
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66/121 thalidomide, thalidomide analogues (such as CC-5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as angiozyme), α interferon (such as interferon a2a), suramine and similar agents), VEGF-R kinase inhibitors and other antiangiogenic tyrosine kinase inhibitors (such as SU011248), specific endothelial survival / integrin signaling inhibitors (such as vitaxin and similar agents), copper chelators / antagonists (such as tetrathiomolybdate, captopril and similar agents), carboxyamido-triazolol (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E as well as angiogenesis-inhibiting nucleotide molecules (such as antisense-VEGF-cDNA, angiostatin-encoding cDNA, p53-encoding cDNA and cDNA defective VEGF receptor-2 encoder).
[00201] Other examples of such angiogenesis, neovascularization, and / or other vascularization inhibitors are antiangiogenic heparin derivatives (eg, heparinase III), temozolomide, NK4, macrophage migration inhibiting factor, cyclooxygenase-2 inhibitors, hypoxia-inducible factor-1 inhibitors, antiangiogenic soy isoflavones, oltipraz, fumagillin and their analogues, somatostatin analogues, pentosan polysulfate, tecogalan sodium, dalteparin, tunstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, canstatin other agents, such as anti-alpha-v / beta-3-integrin and anti-quininostatin antibodies.
[00202] In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody to treat the disorders as described above can be an anti-cancer immunogen, such as cancer antigen / tumor-associated antigen (e.g. , epithelial cell adhesion molecule (EpCAM / TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor-associated glycoprotein-72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1 , MDA7, cancer-associated viral vaccines (eg, human papillomavirus vaccines) or tumor-derived heat shock proteins.
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67/121 [00203] In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody to treat the disorders as described above can be a chemokine, anticancer cytokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (eg, INFa2b), ΙΡΝβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestor , and TNFα. Suitable chemokines include Glu-Leu-Arg (ELR) negative chemokines such as IP-10, MCP-3, MIG, and SDF-Ια from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins. [00204] In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody to treat the disorders as described above can be an apoptosis regulator / cell cycle control (or "regulating agent"). An apoptosis regulator / cell cycle control includes molecules that select and modulate apoptosis regulators / cell cycle control such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that over-stimulate the cell cycle (such as flavopyridol (L868275, HMR1275), 7-hydroxy-staurosporine (UCN-01, KW2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT- 095, GRN163 and compositions described in for example US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with apoptotic routes include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and antisense Bcl-2.
[00205] In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody to treat disorders as described above can be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such agents
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68/121 hormonal regulators are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / stinyl, an antiandrogen (such as flutaminde / eulexin), a progestin (such as hydroxyproxide, progesterone, hydroxyproxide and progesterone) -progesterone / provera, megestrol / megaceus acceptor), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone releasing hormone (and its analogs and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole / arimidex, amino-glutetimide / citraden, exemestane) or a hormone inhibitor (such as octreotide / sandostatin). [00206] In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody to treat disorders as described above can be an antianergic agent, such as compounds that are molecules that block CTLA-4 activity, P. ipilimumab.
[00207] In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody to treat the disorders as described above can be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.
[00208] Examples of other anticancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody to treat the disorders described above are differentiation-inducing agents, analogs of retinoic acid (such as all acids trans-retinoic, 13-cis-retinoic acids and similar agents), vitamin D analogs (such as seocalcitol and similar agents), ErbB3, ErbB4, IGF-IR inhibitors, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, RON (such as an anti-RON antibody), Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.
[00209] Examples of other anticancer agents, which may be relevant as therapeutic agents for use in combination with a
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69/121 anti-c-Met antibody to treat the disorders described above are estramustine and epirubicin.
[00210] Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody to treat the disorders described above are an HSP90 inhibitor such as 17-allyl-amino-geldanamycin, targeted antibodies against a tumor antigen such as PSA, CA125, KSA, integrins, e.g. , β1 integrin, or VCAM inhibitors.
[00211] Examples of other anticancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody to treat the disorders described above are calcineurin inhibitors (such as valspodar, PSC 833 and other MDR inhibitors -1 or p-glycoprotein), TOR inhibitors (such as sirolimus, everolimus and rapamcin), and inhibitors of "lymphocyte-housing" mechanisms (such as FTY720), and agents with effects on cell signaling such as molecule inhibitors. adhesion (eg anti-LFA).
[00212] In one embodiment, the anti-c-Met antibody of the invention is for use in combination with one or more other therapeutic antibodies, such as ofatumumab, zanolimumab, daratumumab, ranibizumab, Zenapax, Simulect, Remicade, Humira, Tysabri, Xolair , raptiva and / or rituximab.
[00213] Other therapeutic antibodies that can be used in combination with the antibody of the present invention are anti-c-Met antibodies that bind to other regions of c-Met, such as the antibodies described in WO2005016382, WO2006015371, WO2007090807, WO2007126799 or WO2009007427 (all incorporated herein by reference).
[00214] In another embodiment, two or more different antibodies of the invention as described herein are used in combination for the treatment of disease. Especially interesting combinations include two or more non-competing antibodies. Such combination therapy can cause
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70/121 binding of an increased number of antibody molecules per cell, which can give increased efficacy, e.g. via complement-mediated lysis activation.
[00215] In addition to the above, other modalities of the combination therapies of the invention include the following:
• For the treatment of non-small cell lung cancer, an anti-c-Met antibody in combination with EGFR inhibitors, such as an anti-EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), or in combination with an ErbB2 inhibitor (Her2 / neu) (such as an anti-HER2 antibody, e.g. trastuzumab, trastuzumab-DM1 or pertuzumab) or in combination with an inhibitor of both EGFR and HER2, such as lapatinib, or in combination with an HER3 inhibitor.
• For the treatment of glioma, an anti-c-Met antibody in combination with temozolomide or an angiogenesis inhibitor, such as bevacizumab.
• For the treatment of colorectal cancer an anti-cMet antibody in combination with one or more compounds selected from: gemcitabine, bevacizumab, FOLFOX, FOLFIRI, XELOX, IFL, oxaliplatin, irinotecan, 5-FU / LV, Capecitabine, UFT, selection agents of EGFR, such as cetuximab. panitumumab, zalutumumab; VEGF inhibitors, or tyrosine kinase inhibitors such as sunitinib.
• For the treatment of prostate cancer an anti-cMet antibody in combination with one or more compounds selected from hormonal / anti-hormonal therapies; such as antiandrogens, luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutic agents such as taxanes, mitoxantrone, estramustine, 5FU, vimblastine, ixabepilone.
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Radiotherapy - surgery [00216] In one embodiment, the present invention provides a method for treating a disorder involving cells expressing c-Met in a subject, the method of which comprises administering a therapeutically effective amount of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, and radiation therapy to a subject in need thereof.
[00217] In one embodiment, the present invention provides a method for treating or preventing cancer, the method of which comprises administering a therapeutically effective amount of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, and radiotherapy to a subject in need of it.
[00218] In one embodiment, the present invention provides the use of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, for the preparation of a pharmaceutical composition for treating cancer to be administered in combination with radiotherapy.
[00219] Radiotherapy may comprise radiation or administration of radiopharmaceuticals associated with a patient is provided. The radiation source can be either external or internal to the patient being treated (radiation treatment can, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used in the practice of such methods include, e.g. e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine131, and indium-111.
[00220] In yet another embodiment, the present invention provides a method for treating or preventing cancer, the method of which comprises administering to a subject in need thereof a therapeutically effective amount of an anti-c-Met antibody, such as an anti -c-Met of the present invention, in combination with surgery.
[00221] Diagnostic uses
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72/121 [00222] The anti-c-Met antibodies of the invention can also be used for diagnostic purposes. Therefore, in a further aspect, the invention relates to a diagnostic composition comprising an anti-c-Met antibody as defined herein.
[00223] In one embodiment, the anti-c-Met antibodies of the present invention can be used in vivo or in vitro to diagnose diseases in which cells activated expressing c-Met play an active role in the pathogenesis, by detecting levels of c- Met, or levels of cells that contain c-Met on their membrane surface. This can be accomplished, for example, by contacting a sample to be tested, optionally together with a control sample, with the anti-c-Met antibody under conditions that allow a complex to form between the antibody and c-Met.
[00224] Thus, in an additional aspect, the invention relates to a method for detecting the presence of c-Met antigen, or a cell expressing c-Met, in a sample comprising:
- contacting the sample with an anti-c-Met antibody of the invention under conditions that allow a complex to form between the antibody and c-Met; and
- analyze whether a complex has been formed.
[00225] In one embodiment, the method is performed in vitro.
[00226] More specifically, the present invention provides methods for the identification of, and diagnosis of, invasive tissues and cells, and other cells selected by anti-c-Met antibodies of the present invention, and for monitoring the progress of therapeutic treatment, of the state after treatment, risk of developing cancer, progression of cancer, and the like.
[00227] Suitable markers for the anti-c-Met antibody and / or secondary antibodies used in such techniques are well known in the art.
[00228] In a further aspect, the invention relates to a kit for
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73/121 detect the presence of c-Met antigen, or a cell expressing c-Met, in a sample comprising
- an anti-c-Met antibody of the invention or a bispecific molecule of the invention; and
- instructions for using the kit.
[00229] In one embodiment, the present invention provides a cancer diagnosis kit comprising a container comprising an anti-c-Met antibody, and one or more reagents for detecting the binding of the anti-c-Met antibody in c-Met. Reagents can include, for example, fluorescent tags, enzyme tags, or other detectable tags. Reagents can also include secondary or tertiary antibodies or reagents for enzyme reactions, with enzyme reactions producing a product that can be visualized.
Anti-idiotypic antibodies [00230] In a further aspect, the invention relates to anti-idiotypic antibody that binds to an anti-c-Met antibody of the invention as described herein.
[00231] An anti-idiotypic antibody (Id) is an antibody that recognizes unique determinants generally associated with the antigen binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and the same type as the source of an anti-c-Met mAb with the mAb for which the anti-Id is being prepared. The immunized animal can typically recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).
[00232] An anti-Id antibody can also be used as an "immunogen" to induce an immune response in yet another animal, producing an so-called anti-anti-Id antibody. An anti-anti-Id antibody may be epitopically identical to the original mAb, which
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74/121 induced the anti-Id antibody. Thus, by using antibodies for the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificities.
[00233] The present invention is hereinafter illustrated by the following examples which are not to be understood as additional limitations.
EXAMPLES
Example 1: Expression constructs for c-Met [00234] Codon-optimized constructs were generated for expression of c-Met, the extracellular domain (ECD) (aa 1- 932 and a His6 Cterminal tag or the SEMA domain of c-Met (aa 1-567 and a His9 Cterminal tag), in HEK or CHO cells. The proteins encoded by these constructs are identical to the Gen Bank Accession Number NM 000245 for c-Met. The constructs contained restriction sites suitable for cloning and an optimal Kozak sequence (Kozak et al. (1999) Gene 234: 187208). The constructs were cloned into the mammalian expression vector pEE13.4 (Lonza Biologies) (Bebbington (1992) Biotechnology (NY) 10: 169175), obtaining pEE13.4cMet, pEE13.4cMetECDHis and pEE13.4cMetSEMA567His8.
Example 2: Expression constructs for 5D5v1, 5D5 and G11-HZ [00235] Codon-optimized constructs for expression of the heavy chain (HC) and light chain (LC) of IgG1 antibodies were generated: 5D5vl, 5D5 and G11-HZ in HEK cells. The proteins encoded by these constructs are identical to those described in US patent 6468529 (sequence numbers 3 and 4) for heavy chain and light chain 5D5v1, WO 2006/015371 A2 (fig. 13) for heavy chain and light chain 5D5 and WO 2009 / 007427 A2 (sequence was extracted from multiple figures) for 224G11 heavy and light chains. 224G11 is also called G11-HZ.
Example 3: Transient expression in HEK-293F cells
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75/121 [00236] Freestyle ™ 293-F cells (a HEK-293 subclone adapted for growth in suspension and chemically defined Freestyle medium (HEK-293F)) were obtained from Invitrogen and transfected with appropriate plasmid DNA using 293fectin (Invitrogen) according to the manufacturer's instructions. Expression of c-Met was tested by means of analysis using FACS as described below. In the case of antibody expression, the appropriate heavy and light chain expression vectors were coexpressed.
Example 4: Transient expression in CHO cells [00237] pEE13.4cMet was transiently transfected into Freestyle ™ CHO-S cell line (Invitrogen) using a Freestyle MAX transfection reagent (Invitrogen). Expression of c-Met was tested by analysis using FACS as described below.
Example 5: Cloning and expression of monovalent antibodies (UniBody® molecules) [00238] For the expression of monovalent antibodies in mammalian cells the HC constant region of IgG4, missing from the hinge region (Ch) (amino acids E99-P110) and containing 2 mutations F405T and Y407E in the CH3 region, it was synthesized as a codon-optimized construct in pcDNA3.3 mammalian expression vector (Invitrogen) and called pUniTE. A separate vector was constructed by inserting the codon optimized constant region in the aqueous dispersion region of the human kappa light chain into pcDNA3.3 and called pKappa.
[00239] Relevant VH and VL regions were inserted into pUniTE and pKappa respectively resulting in vectors for the expression of the heavy and light chains of specific antibodies. Cotransfection of the heavy and light chain vectors of a specific antibody in HEK-293F cells (Invitrogen), resulted in the transient production of monovalent antibodies with the desired specificities. Purification was performed using chromatography
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76/121 in Protein A affinity column (as described in Example 11).
Example 6: Purification of His-tagged c-Met [00240] cMetECDHis and cMetSEMAHis were expressed in HEK-293F cells. The His-tag on cMetECDHis and cMetSEMAHis allows purification by immobilized metal affinity chromatography. In this process, a chelator fixed on the chromatographic resin is loaded with Co ²⁺ cations. Supernatants containing cMetECDHis and cMetSEMAHis were incubated with the resin in batch mode (ie solution). The His-tagged protein binds strongly on the resin beads, while other proteins present in the culture supernatant do not bind strongly. After incubation, the beads are recovered from the supernatant and packed into a column. The column is washed for the purpose of removing loosely bound proteins. The tightly bound cMetECDHis and cMetSEMAHis proteins are then eluted with a buffer containing imidazole, which competes with the binding of His in Co ²⁺ . The eluent is removed from the protein by buffer exchange on a desalination column.
Example 7: Immunization procedure of transgenic mice [00241] Antibodies 005, 006, 007, 008, 011, 012, 016, 017, 022, 024, 025, 028, 031, 035, 039, 040, 045, 093, 095 , 096, 101 and 104 were derived from the following immunizations: one HCo20 mouse (1 female, GG2713 strain), one HCo17 mouse (female, GG2714 strain) and two HCo12Balb / C mice (2 females, GG2811 strain) (Medarex, San Jose , CA, USA; for references see above paragraph on HuMab mouse, WO2009097006 and US2005191293) were immunized every fortnight alternating with 5x10 ⁶ NCI-H441 tumor cells intraperitoneally (IP) and 20 pg of cMetECDHis protein copulated with the keyhole limpet hapten “ keyhole ”[Diodora aspera] (KLH) subcutaneously (SC).
[00242] Antibodies 058, 061, 062, 063, 064, 065, 066, 068, 069, 078,
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082, 084, 087, 089, 098 and 181 were derived from the following immunizations: two HCo20 mice (1 male and 1 female, strain GG2713) and one HCo12-Balb / C mouse (1 male, strain GG2811) (Medarex, San Jose , CA, USA; for references see above paragraph on HuMab mouse) were immunized every fortnight alternating with 5x106 CHOK1SV cells transiently transfected with cMetECD intraperitoneally (IP) and 20 gg of cMetECDHis protein copulated in "keyhole" key hemocyanine hapten ( KLH) subcutaneously (SC).
[00243] A maximum of eight immunizations were performed per mouse, four IP immunizations and four SC immunizations at the base of the tail. The first cell immunization was done in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other immunizations, cells were injected IP into PBS and cMetECD copulated with KLH was injected SC using incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). Mice with at least two sequential c-Met antibody titers of 200 (1/200 serum dilutions) or higher, detected in the FMAT antigen specific selection assay as described in Example 8, had splenocyte and lymph node cells fused into a mouse myeloma cell line.
Example 8: Specific homogeneous antigen selection assay [00244] The presence of anti-c-Met antibodies in immunized mouse sera or HuMab transfectome or hybridoma culture supernatant (human monoclonal antibody) was determined by specific selection assays of homogeneous antigen (four quadrants) using Fluorometric Microvolumetric Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA). For this, a combination of 3 cell-based assays and an account-based assay was used.
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In cell-based assays, binding on TH1016-cMet (HEK-293F cell transiently expressing the extracellular domain of the c-Met receptor; produced as described above) and HT29 cell (which expresses c-Met on the cell surface) was determined as well as in wild type HEK293 cells (negative control that does not express c-Met). For the bead-based assay, binding in SB1016-cMet (cMetECDHis obtained from transiently transfected HEK-293F cells as described above, biotinylated and copulated into streptavidin-coated beads) was determined. Samples were added to the cells / beads to allow c-Met binding. Subsequently, HuMab binding was detected using a fluorescent conjugate (Goat anti-human IgG Cy5 antibody, Jackson ImmunoResearch). The chimeric antibody 5D5v1 specific for c-Met (produced in HEK-293F cells) was used as a positive control and the pooled mouse serum-HuMab and HuMab-KLH were used as negative controls. The samples were scanned using an Applied Biosystems 8200 Cellular Detection System (8200 CDS) and ‘counts x fluorescence’ was used as a reading. Samples were classified as positive when counts were over 50 and counts x fluorescence were at least three times higher than the negative HuMab-KLH control.
Example 9: Generation of HuMab hybridoma [00245] HuMab mice with the development of a specific titer for sufficient antigen (as defined above) were killed and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocyte cells and lymph nodes into a mouse myeloma cell line was performed by electrofusion using an CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially according to the manufacturer's instructions. Fusion plates were selected with the specific antigen binding assay
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79/121 as described above and the positives from this assay were tested in an Alphascreen® SureFire® ERK-phosphorylation assay and affinity rating Octet assay as described below. Antibodies 031, 035, 087 and 089 have been expanded and grown based on standard protocols (eg, as described in Coligan JE, Bierer, BE, Margulies, DH, Shevach, EM and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).
[00246] In parallel antibodies 005, 006, 007, 008, 011, 012, 016, 017, 022, 024, 025, 028, 035, 039, 040, 045, 058, 061, 062, 063, 064, 065, 066, 068, 069, 078, 082, 084, 093, 095, 096, 098, 101, 104 and 181 were cloned using the ClonePix system (Genetix, Hampshire, United Kingdom). Specific primary cavity hybridomas were seeded on a solid medium made of 40% CloneMedia (Genetix, Hampshire, UK) and 60% complete HyQ 2x medium (Hyclone, Waltham, USA) and approximately 100 primary hybridoma subclones of the cavity were selected . The subclones were retested in the antigen specific binding assay as previously described and IgG levels were measured using Octet in order to select the best producer and specific clone for primary cavity hybridoma for further expansion. Further expansion and culture of the resulting HuMab hybridomas was done based on standard protocols (eg, as described in Coligan JE, Bierer, BE, Margulies, DH, Shevach, EM and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).
Example 10: Purified antibody mass spectrometry [00247] Small 0.8 mL aliquots of hybridoma supernatant containing Hyperflask stage antibody or 6 wells were purified using PhyTip columns containing Protein G resin (PhyNexus Inc., San Jose, USA) on a Sciclone ALH 3000 workstation (Caliper
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Lifesciences, Hopkinton, USA). PhyTip columns were used according to the manufacturer's instructions, but the buffers were replaced by: PBS Binding Buffer (B. Braun, Medical BV, Oss, Netherlands) and Glycine-HCl 0.1 M pH Elution Buffer 2.7 (Fluka Riedel-de Haen, Buchs, Germany). After purification, the samples were neutralized with 2 M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases larger volumes of culture supernatant were purified using Protein A affinity column chromatography.
[00248] After purification, the samples were applied to a 384 well plate (Waters, 100 pL square well plate, part number 186002631). The samples were deglycosylated overnight at 37 ° C with N-glycosidase F. DTT (15 mg / ml) was added (1 pL / well) and incubated for 1 h at 37 ° C. The samples (5 or 6 pL) were desalted in an Acquity UPLC ™ (Waters, Milford, USA) with a BEH300 C18 column, 1.7 pm, 2.1 mm x 50 mm at 60 ° C. MQ water and LC-MS grade acetonitrile (Biosolve, catalog number 01204101, Valkenswaard, Netherlands) both with 0.1% formic acid (Fluka, catalog number 56302, Buchs, Germany), were used as Eluents A and B, respectively. Flight time mass spectra with electron spray ionization were recorded on a micrOTOF ™ mass spectrometer (Bruker, Bremen, Germany) operating in positive ion mode. Before analysis, a 900-3,000 m / z scale was calibrated with an ES adjustment mixture (Agilent Technologies, Santa Clara, USA). Mass spectra were subjected to deconvolution by DataAnalysis ™ software v. 3.4 (Bruker) using the search with Maximal Entropy algorithm for molecular weights between 5 and 80 kDa.
[00249] After deconvolution, the masses of heavy and light chains for all samples were compared for the purpose of finding duplicate antibodies. When comparing heavy chains, the possible presence of C-terminal lysine variants was taken into account. This resulted in
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81/121 a list of singular antibodies, in which singular is defined as a singular combination of heavy and light chains. In case duplicate antibodies were found, the results of the other tests were used to decide which antibody was the best material to continue with the experiments.
Example 11: Sequence analysis of the anti-c-Met antibody variable domains and cloning into expression vectors [00250] HuMabs total anti-c-Met RNA was prepared from 5x106 hybridoma cells and 5'-RACE-Complementary DNA ( cDNA) was prepared from 100 ng of total RNA, using the SMART RACE cDNA Amplification kit (Clontech), according to the manufacturer's instructions. Coding regions for VH (heavy chain variable region) and VL (light chain variable region) were amplified by PCR and cloned into matrix in the pG1f constant region vectors (containing the constant, fully synthetic, codon-optimized heavy chain region) of human IgG1 (allotype f) in the mammalian expression vector pEE6.4 (Lonza Biologies, Slough, United Kingdom (Bebbington et al. (1992) Biotechnology 10: 169-175)) and pKappa (containing the constant region, totally synthetic, codon-optimized human kappa light chain (allotype Km3) in the mammalian expression vector pEE12.4 (Lonza Biologies, Slough, United Kingdom (Bebbington et al. (1992) Biotechnology 10: 169-175)) using a independent binding cloning strategy (Aslanidis et al. 1990 Nucleic Acids Res. 18: 60696074). For each HuMab, 12 VL clones and 8 VH clones were sequenced and their theoretical masses were calculated and compared with the mass spectrometry data of antibody disp The sequences are shown in the Sequence Listing and in Table 1 below. CDR sequences are defined according to Kabat et al., “Sequences of Proteins of Immunological Interest”, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Table 2 and Table 3 give an
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82/121 overview of the most homologous antibody sequence and germline sequences.
Table 1: Heavy chain variable region (VH), light chain variable region (VL) and CDR sequences from HuMabs SEQ ID No: 1 VH 005 QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYGFGWVRQAPGQGLEWMGRISPILGIA NYAQMFQGRVTITADKSTSTAYMELSSLR SEDTAVYYCARDVGYDWPDTFDIWGQGT MVIVSS SEQ ID No: 2 VH 005, CDR1 SYGFG SEQ ID No: 3 VH 005, CDR2 RISPILGIANYAQMFQG SEQ ID No: 4 VH 005, CDR3 DVGYDWPDTFDI SEQ ID No: 5 VL 005 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGGGSGTDFTLTISSLQPEDFATYYCQQ YNSFPPTFGQGTKVEIK SEQ ID No: 6 VL 005, CDR1 RASQGISSWLA SEQ ID No: 7 VL 005, CDR2 AASSLQS SEQ ID No: 8 VL 005, CDR3 QQYNSFPPT SEQ ID No: 9 VH 006 QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSFGIGWVRQAPGQGLEWMGRIFPILGTAN YAQMFQGRVTITADKSTSTAYMELTSLRS EDTAVYYCARDVGYDSADAFDIWGQGTM VTVSS SEQ ID No: 10 VH 006, CDR1 SFGIG SEQ ID No: 11 VH 006, CDR2 RIFPILGTANYAQMFQG SEQ ID No: 12 VH 006, CDR3 DVGYDSADAFDI SEQ ID No: 13 VL 006 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQY NSYPPTFGQGTKVEIK SEQ ID No: 14 VL 006, CDR1 RASQGISSWLA SEQ ID No: 15 VL 006, CDR2 AASSLQS SEQ ID No: 16 VL 006, CDR3 QQYNSYPPT SEQ ID No: 17 VH 008 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSETR YSPSFQGQVTISADKSISTAYLQWSSLKAS DTAMYYCARQEITGEFDYWGQGTLVTVSS SEQ ID No: 18 VH 008, CDR1 SYWIG SEQ ID No: 19 VH 008, CDR2 IIYPGDSETRYSPSFQG SEQ ID No: 20 VH 008, CDR3 QEITGEFDY
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SEQ ID No: 21 VL 008 AIQLTQSPSSLSASVGDRVTITCRASQGISS ALAWYQQKPGKAPKLLIYDASSLESGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQF NSYPRTFGQGTKVEIK SEQ ID No: 22 VL 008, CDR1 RASQGISSALA SEQ ID No: 23 VL 008, CDR2 DASSLES SEQ ID No: 24 VL 008, CDR3 QQFNSYPRT SEQ ID No: 25 VH 022 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMHWVRQAPGKGLEWVAVISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARELLWFGELWGYFDLWGR GTLVTVSS SEQ ID No: 26 VH 022, CDR1 SYAMH SEQ ID No: 27 VH 022, CDR2 VISYDGSNKYYADSVKG SEQ ID No: 28 VH 022, CDR3 ELLWFGELWGYFDL SEQ ID No: 29 VL 022 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQEA SSFTWTFGQGTKVEIK SEQ ID No: 30 VL 022, CDR1 RASQGISSWLA SEQ ID No: 31 VL 022, CDR2 AASSLQS SEQ ID No: 32 VL 022, CDR3 QEASSFTWT SEQ ID No: 33 VH 024 EVQLLESGGGLVQPGGSLRLSCVASGFTFS SYAMSWVRQAPGKGLEWVSAISGSSGGST YYVDSVKGRFTISRANSKNTLYLQMNSLR AEDTAVYYCAKDLDRGWMGYFGYWGQG TLVTVSS SEQ ID No: 34 VH 024, CDR1 SYAMS SEQ ID No: 35 VH 024, CDR2 AISGSSGGSTYYVDSVKG SEQ ID No: 36 VH 024, CDR3 DLDRGWMGYFGY SEQ ID No: 37 VL 024 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPTFGQGTRLEIK SEQ ID No: 38 VL 024, CDR1 RASQGISSWLA SEQ ID No: 39 VL 024, CDR2 AASSLQS SEQ ID No: 40 VL 024, CDR3 QQANSFPT SEQ ID No: 41 VH 035 EVQLVQSGAEVKKPGESLKISCKGSGYSFT SYWIGWVRQMPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTAYLQWNSLKA SDTAMYYCARQEITGEFDYWGQGTLVTVS S SEQ ID No: 42 VH 035, CDR1 SYWIG SEQ ID No: 43 VH 035, CDR2 IIYPGDSDTRYSPSFQG SEQ ID No: 44 VH 035, CDR3 QEITGEFDY
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SEQ ID No: 45 VL 035 AIQLTQSPSSLSASVGDRVTITCRASQGISS ALAWYQQKPGKAPKLLIYDASSLESGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQF NSYPMYTFGQGTKLEIK SEQ ID No: 46 VL 035, CDR1 RASQGISSALA SEQ ID No: 47 VL 035, CDR2 DASSLES SEQ ID No: 48 VL 035, CDR3 QQFNSYPMYT SEQ ID No: 49 VH 045 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWVSVISGSGGITY YADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARDRGWGSDYWGQGTLVTVS S SEQ ID No: 50 VH 045, CDR1 SYAMS SEQ ID No: 51 VH 045, CDR2 VISGSGGITYYADSVKG SEQ ID No: 52 VH 045, CDR3 DRGWGSDY SEQ ID No: 53 VL 045 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQR SNWPFTFGPGTKVDIK SEQ ID No: 54 VL 045, CDR1 RASQSVSSYLA SEQ ID No: 55 VL 045, CDR2 DASNRAT SEQ ID No: 56 VL 045, CDR3 QQRSNWPFT SEQ ID No: 57 VH 058 EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDDGSYT YYPDSVKGRFTISRDNAKNNLYLQMSSLK SEDTAMYYCAREGLYYYGSGSYYNQDYW GQGTLVTVSS SEQ ID No: 58 VH 058, CDR1 DYYMY SEQ ID No: 59 VH 058, CDR2 TISDDGSYTYYPDSVKG SEQ ID No: 60 VH 058, CDR3 EGLYYYGSGSYYNQDY SEQ ID No: 61 VL 058 AIQLTQSPSSLSASVGDRVTITCRASQGLSS ALAWYRQKPGKAPKLLIYDASSLESGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQF TSYPQITFGQGTRLEIK SEQ ID No: 62 VL 058, CDR1 RASQGLSSALA SEQ ID No: 63 VL 058, CDR2 DASSLES SEQ ID No: 64 VL 058, CDR3 QQFTSYPQIT SEQ ID No: 65 VH 061 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGX1IYHSGNT YDNPSLKSRVTIAVDRSKNQLSLKLSFLTA ADTAVYYCARSSYDFLTDWGQGTLVTVS, or, Y, ANY, YES, whatever, X1 SEQ ID No: 66 VH 061, CDR1 SGGHSWS
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SEQ ID No: 67 VH 061, CDR2 X1IYHSGNTYDNPSLKS, where X1 is any amino acid, preferably C, S, Y or A SEQ ID No: 68 VH 061, CDR3 SSYDFLTD SEQ ID No: 69 VL 061 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NGFPITFGQGTRLEIK SEQ ID No: 70 VL 061, CDR1 RASQGISSWLA SEQ ID No: 71 VL 061, CDR2 AASSLQS SEQ ID No: 72 VL 061, CDR3 QQANGFPIT SEQ ID No: 73 VH 062 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGX1IYHSGNT YDNPSLKSRVTIAVDRSKNQLSLKLSFVTA ADTAVYYCARSSYDILTDWGQGTLVTVSSYE, YES, RIGHT, YES, RIGHT, YES, RIGHT, YES SEQ ID No: 74 VH 062, CDR1 SGGHSWS SEQ ID No: 75 VH 062, CDR2 X1IYHSGNTYDNPSLKS, where X1 is any amino acid, preferably C, S, Y or A SEQ ID No: 76 VH 062, CDR3 SSYDILTD SEQ ID No: 77 VL 062 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NGFPITFGQGTRLEIK SEQ ID No: 78 VL 062, CDR1 RASQGISSWLA SEQ ID No: 79 VL 062, CDR2 AASSLQS SEQ ID No: 80 VL 062, CDR3 QQANGFPIT SEQ ID No: 81 VH 064 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGX 1 IYHSGNT YDNPSLKSRVTISVDRSKNQVSLKLSSVTA ADTAVYYCARSSYDILTDWGQGTLVTVSS YY, anytime, anytime, anytime, anytime, whatever, X1 SEQ ID No: 82 VH 064, CDR1 SGGHSWS SEQ ID No: 83 VH 064, CDR2 X1IYHSGNTYDNPSLKS, where X1 is any amino acid, preferably C, S, Y or A SEQ ID No: 84 VH 064, CDR3 SSYDILTD SEQ ID No: 85 VL 064 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NGFPITFGQGTRLEIK SEQ ID No: 86 VL 064, CDR1 RASQGISSWLA SEQ ID No: 87 VL 064, CDR2 AASSLQS
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SEQ ID No: 88 VL 064, CDR3 QQANGFPIT SEQ ID No: 89 VH 068 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGYSWSWIRQPPGKGLEWIGX1IYHSGSTY YNPSLKSRVTISVDRSKNQFSLKLSSVTAA DTAVYYCARSSYDILTDWGQGTLVTVSS YY, Either, whatever, X1 SEQ ID No: 90 VH 068, CDR1 SGGYSWS SEQ ID No: 91 VH 068, CDR2 X1IYHSGSTYYNPSLKS, where X1 is any amino acid, preferably C, S, Y or A SEQ ID No: 92 VH 068, CDR3 SSYDILTD SEQ ID No: 93 VL 068 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 94 VL 068, CDR1 RASQGISSWLA SEQ ID No: 95 VL 068, CDR2 AASSLQS SEQ ID No: 96 VL 068, CDR3 QQANSFPIT SEQ ID No: 97 VH 069 QVQLVQSGAEVKKPGASVKVSCETSGYTF TSYGISWVRQAPGHGLEWMGWISAYNGY TNYAQKLQGRVTMTTDTSTSTAYMELRSL RSDDTAVYYCARDLRGTNYFDYWGQGTL VTVSS SEQ ID No: 98 VH 069, CDR1 SYGIS SEQ ID No: 99 VH 069, CDR2 WISAYNGYTNYAQKLQG SEQ ID No: 100 VH 069, CDR3 DLRGTNYFDY SEQ ID No: 101 VL 069 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWFQHKPGKAPKLLIYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 102 VL 069, CDR1 RASQGISNWLA SEQ ID No: 103 VL 069, CDR2 AASSLLS SEQ ID No: 104 VL 069, CDR3 QQANSFPIT SEQ ID No: 105 VH 096 EVQLVQSGAEVKKPGESLKISCKGSGYSFT SYWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEITGDFDYWGQGTLVTVSS SEQ ID No: 106 VH 096, CDR1 SYWIG SEQ ID No: 107 VH 096, CDR2 IIYPGDSDTRYSPSFQG SEQ ID No: 108 VH 096, CDR3 QEITGDFDY SEQ ID No: 109 VL 096 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPNLLIYAASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNS YPLTFGGGTKVEIK
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SEQ ID No: 110 VL 096, CDR1 RASQGISSALA SEQ ID No: 111 VL 096, CDR2 AASSLES SEQ ID No: 112 VL 096, CDR3 QQFNSYPLT SEQ ID No: 113 VH 098 QVQLVQSGAEVKKPGASVKVSCKASGYTF TNFGISWVRQAPGQGLEWMGWISAFNGHT DYSQKVQGRVTMTTDTSTSTAYMELRSLR SDDTAVFYCARSHYYGSGSPFDYWGQGTL VTVSS SEQ ID No: 114 VH 098, CDR1 NFGIS SEQ ID No: 115 VH 098, CDR2 WISAFNGHTDYSQKVQG SEQ ID No: 116 VH 098, CDR3 SHYYGSGSPFDY SEQ ID No: 117 VL 098 DIQMTQSPSSLSASVGDRVTITCRASQGISN WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCHQY KSYPWTFGQGTKVEIK SEQ ID No: 118 VL 098, CDR1 RASQGISNWLA SEQ ID No: 119 VL 098, CDR2 AASSLQS SEQ ID No: 120 VL 098, CDR3 HQYKSYPWT SEQ ID No: 121 VH 101 QVQLVQSGGEVKKPGASVKVSCKASGYTFTRHGITWVRQAPGQGLEWMGWISADNGN TNYAQKFQDRVTMTTDTSTSTAYMELRSL RSDDTAVYFCARVFRYFDWLLPYFDYWG QGTLVTVST SEQ ID No: 122 VH 101, CDR1 RHGIT SEQ ID No: 123 VH 101, CDR2 WISADNGNTNYAQKFQD SEQ ID No: 124 VH 101, CDR3 VFRYFDWLLPYFDY SEQ ID No: 125 VL 101 EIVLTQSPGTLSLSPGERATLSCRASQSVSSS YLAWYQQKPGQAPRLLIYGVFSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSSPYTFGQGTKLEIK SEQ ID No: 126 VL 101, CDR1 RASQSVSSSYLA SEQ ID No: 127 VL 101, CDR2 GVFSRAT SEQ ID No: 128 VL 101, CDR3 QQYGSSPYT SEQ ID No: 129 VH 181 QVQLVQSGAEVKKPGASVKVSCKASGYTF TSYGISWVRQAPGQGLEWMGWISTYNGYT NYAQKLQGRVTMTTDTSTSTAYMELRSLR SDDTAVYYCARDLRGTAYFDYWGQGTLV TVSS SEQ ID No: 130 VH 181, CDR1 SYGIS SEQ ID No: 131 VH 181, CDR2 WISTYNGYTNYAQKLQG SEQ ID No: 132 VH 181, CDR3 DLRGTAYFDY SEQ ID No: 133 VL 181 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK
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SEQ ID No: 134 VL 181, CDR1 RASQGISNWLA SEQ ID No: 135 VL 181, CDR2 AASSLLS SEQ ID No: 136 VL 181, CDR3 QQANSFPIT SEQ ID No: 137 VH 066 QVQLVQSGAEVKKPGASVKVSCEASGYTF TSYGISWVRQAPGHGLEWMGWISAYNGY TNYAQKLQGRVTMTADTSTSTAYMELRSL RSDDTAVYYCARDLRGTNYFDYWGQGTL VTVSS SEQ ID No: 138 VL 066 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 139 VH 065 QVQLVQSGAEVKKPGASVKVSCEASGYTF TNYGISWVRQAPGHGLEWMGWISAYNGY TNYAQKLQGRVTMTTDTSTTTAYMELRSL RSDDTAVYYCARDLRGTNYFDYWGQGTL VTVSS SEQ ID No: 140 VL 065 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 141 VH 082 QVQLVQSGAEVKKPGASVKVSCETSGYTF TSYGISWVRQAPGHGLEWMGWISAYNGY TNYAQKLQGRVTMTTDTSTSTAYMELRSL RSDDTAVYYCARDLRGTNYFDYWGQGTL VTVSS SEQ ID No: 142 VL 082 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 143 VH 089 QVQLVQSGAEVKKPGASVKVSCETSGYTF TSYGISWVRQAPGHGLEWMGWISAYNGY TNYAQKLQGRVTMTTDTSTSTAYMELRSL RSDDTAVYYCARDLRGTNYFDYWGQGTL VTVSS SEQ ID No: 144 VL 089 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWFQHKPGKAPKLLIYAASSLLSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 145 VH 031 QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYGFGWVRQAPGQGLEWMGRISPILGITN YAQMFQGRVTITADKSTSTAYMELSSLRSE DTAVYYCARDVGYDWPDTFDIWGQGTMV IVSS SEQ ID No: 146 VL 031 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGGGSGTDFTLTISSLQPEDFATYYCQQ YNSFPPTFGQGTKVEIK
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SEQ ID No: 147 VH 007 QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYGIGWVRQAPGQGLEWMGRIFPILGTAN YAQMFQGRVTITADKSTSTAYIELTSLRSE DTAVYYCARDVGYDSADAFDIWGQGTMV TVSS SEQ ID No: 148 VL 007 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQY NSYPPTFGQGTKVEIK SEQ ID No: 149 VH 011 QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYGIGWVRQAPGQGLEWMGRVFPILGTA NYAQMFQGRVTITADKSTSTAYMELTSLR SEDTAVYYCARDVGYDSADAFDIWGQGT MVTVSS SEQ ID No: 150 VL 011 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQY NSYPPTFGQGTKVEIK SEQ ID No: 151 VH 017 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMHWVRQAPGKGLEWVAFISYDGSNK YFADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARELLWFGELWGYFDLWGRG TLVTVSS SEQ ID No: 152 VL 017 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQEA NSFTWTFGQGTKVEIK SEQ ID No: 153 VH 025 QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMHWVRQAPGKGLEWVAFISYDGSSK DYADSVKGRFTIFRDNSKNTLYLQMSSLRA ADTAVYYCARELLWFGELWGYFDLWGRG TLVTVSS SEQ ID No: 154 VL 025 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQT NSFTWTFGQGTKVEIK SEQ ID No: 155 VH 040 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMTWVRQAPGKGLEWVSVISGSGGITY YADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARDRGWGSDYWGQGTLVTVS S SEQ ID No: 156 VL 040 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQR SNWPFTFGPGTKVDIK SEQ ID No: 157 VH 039 EVQLLESGGGLVQPGGSLRLSCAASGFTFN NYAMSWVRQAPGKGLEWVSAISGSGGITY YADSEKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKDRGWGSDCWGQGTLVTVSS
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SEQ ID No: 158 VL 039 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQR SNWPFTFGPGTKVDIK SEQ ID No: 159 VH 078 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGCLYHSGNTY YNPSLKSRVTISVDRSKNQFSLKLSSVTAA DTAVYYCARSSYDILTDWGQGILVTVSS SEQ ID No: 160 VL 078 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 161 VH 084 QLQLQESGSGLVKPSQTLSLTCGVSGGSISS GGHSWSWIRQPPGKGLEWIGCLYHSGNTY YNPSLKSRVTISVDRSKNQFSLKLSSVTAA DTAVYYCARSSYDILTDWGQGTLVTVSS SEQ ID No: 162 VL 084 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NSFPITFGQGTRLEIK SEQ ID No: 163 VH 063 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGCIYHSGNTY DNPSLKSRVTIAVDRSKNQLSLKLSFVTAA DTAVYYCARSSYDILTDWGQGTLVTVSS SEQ ID No: 164 VL 063 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NGFPITFGQGTRLEIK SEQ ID No: 165 VH 087 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGCIYHSGNTY DNPSLKSRVTISVDRSKNQFSLKLSSVTAA DTAVYYCARSSYDILTDWGQGTLVTVSS SEQ ID No: 166 VL 087 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQA NGFPITFGQGTRLEIK SEQ ID No: 167 VH 016 EVQLVQSGAEVKKPGESLKISCKGSGYIFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEVTGDFDYWGQGTLVTVSS SEQ ID No: 168 VL 016 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNS YPLTFGGGTKVEIK
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SEQ ID No: 169 VH 028 EVQLVQSGGEVKKPGESLKISCKGSGYSFT SYWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEVTGDFDYWGQGTLVTVSS SEQ ID No: 170 VL 028 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNS YPLTFGGGTKVEIK SEQ ID No: 171 VH 012 EVQLVQSGAEVKKPGESLKISCKGSGYSFT SYWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEITGEFDYWGQGTLVTVSS SEQ ID No: 172 VL 012 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNS YPRTFGQGTKVEIK SEQ ID No: 173 VH 095 EVQLVQSGAEVKKPGESLKISCKGSGYSFT SYWIGWVRQMPGKGLEWMGIIYPGDSNTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEITGDFDYWGQGTLVTVSS SEQ ID No: 174 VL 095 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNS YPLTFGGGTKVEIK SEQ ID No: 175 VH 093 EVQLVQSGAEVKKPGESLKISCKGSGYSFT SYWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEITGDFDYWGQGTLVTVSS SEQ ID No: 176 VL 093 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPNLLIYAASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNS YPLTFGGGTKVEIK SEQ ID No: 177 VH 104 EVQLVQSGAEVKKPGESLKISCKGSGYSFIS YWIGWVRQMPGKGLEWMGIIYPGDSDTR YSPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARQEITGDFDYWGQGTLVTVSS SEQ ID No: 178 VL 104 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYVASSLESGVPSRF SGSGSGTDFTLTITSLQPEDFATYYCQQFNS YPITFGQGTRLEIK
Table 2: Homologies of heavy chain and mouse origin sequences Antibody: number ofmouse: strain ofmouse: Germline VH:
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TH1016-005 339732 HCo12B, C1 IgHV1-69-4 TH1016-006 339732 HCo12B, C1 IgHV1-69-4 TH1016-008 339732 HCo12B, C1 IgHV5-51-1 TH1016-022 339733 HCo12B, C1 IgHV3-30-3 * 1 TH1016-024 339733 HCo12B, C1 IgHV3-23-1 TH1016-035-D09 339732 HCo12B, C1 IgHV5-51-1 TH1016-045 339282 HCo17, C1 IgHV3-23-1 TH1016-058 343191 HCo12B, C2 IgHV3-11-3 TH1016-061 348072 HCo20, C2 IgHV4-30-2 * 1 TH1016-062 348072 HCo20, C2 IgHV4-30-2 * 1 TH1016-064 348072 HCo20, C2 IgHV4-30-2 * 1 TH1016-068 348072 HCo20, C2 IgHV4-30-2 * 1 TH1016-069 348072 HCo20, C2 IgHV1-18-1 TH1016-096 339732 HCo12B, C1 IgHV5-51-1 TH1016-098 347330 HCo20, C2 IgHV1-18-1 TH1016-101 340659 HCo20, C1 IgHV1-18-1 TH1016-181 348072 HCo20, C2 IgHV1-18-1
Table 3: Homologies of light chain and mouse origin sequences Antibody: number ofmouse: strain ofmouse: Germline VL: PC1016-005 339732 HCo12B, C1 IGKV1D-16 * 01 PC1016-006 339732 HCo12B, C1 IGKV1D-16 * 01 PC1016-008 339732 HCo12B, C1 IGKV1-13 * 02 PC1016-022 339733 HCo12B, C1 IGKV1-12 * 01 PC1016-024 339733 HCo12B, C1 IGKV1-12 * 01 P1016-035 339732 HCo12B, C1 IGKV1-13 * 02 PC1016-045 339282 HCo17, C1 IGKV3-11 * 01 PC1016-058 343191 HCo12B, C2 IGKV1-13 * 02 PC1016-061 348072 HCo20, C2 IGKV1-12 * 01 PC1016-062 348072 HCo20, C2 IGKV1-12 * 01 PC1016-064 348072 HCo20, C2 IGKV1-12 * 01 PC1016-068 348072 HCo20, C2 IGKV1-12 * 01 PC1016-069 348072 HCo20, C2 IGKV1-12 * 01 PC1016-096 339732 HCo12B, C1 IGKV1-13 * 02 PC1016-098 347330 HCo20, C2 IGKV1D-16 * 01 PC1016-101 340659 HCo20, C1 IGKV3-20 * 01 PC1016-181 348072 HCo20, C2 IGKV1-12 * 01
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93/121 [00251] Figures 1 and 2 show an alignment of HuMabs sequences. Based on these sequences, the consensus sequence can be defined for some of the CDR sequences. These consensus strings are shown in Table 4.
Table 4: Consensus strings SEQ ID No:179 005-006 IgHV1-69-4 CDR1 SX1X2X3X4 X1 = Y or F, X2 = A or G, X3 = F or I, X4 = S or G. Preferably, X1 = Y or F, X2 = G, X3 = For I and X4 = G. SEQ ID No:180 005-006 IgHV1-69-4 CDR2 RX1X2PILGX3X4NYAQX5FQG where X1 = I or V, X2 = I, S or F, X3 = I or T, X4 = A or T, X5 = K or M. Preferably,where X1 = I or V, X2 = S or F, X3 = I or T, X4 = A or T and X5 = M. SEQ ID No:181 005-006 IgHV1-69-4 CDR3 DVGYDX1X2DX3FDI where X1 = W or S, X2 = P or A, X3 = T or A SEQ ID No:182 008-035 IgHV5-51-1 CDR2 IIYPGDSXITRYSPSFHQ where X1 = D, E or N SEQ ID No:183 008-035-096 IgHV5-51-1 CDR3 QEX1TGX2FDY where X1 = V or I, X2= E or D SEQ ID No:184 022 IgHV3-30-3 * 1 CDR2 X1ISYDGSX2KX3X4ADSVKG where X1 = V or F, X2 = N or S, X3 = D or Y, X4 = Y or F SEQ ID No:185 024 IgHV3-23-1 CDR2 AISGSX1GGSTYYX2DSVKG where X1 = S or aa, X2 = V or A SEQ ID No:186 045 IgHV3-23-1 CDR1 Χ1ΥΑΜΧ2 where X1 = Y or N,X2 = S or T SEQ ID No:187 045 IgHV3-23-1 CDR2 X1ISGSGGX2TYYADSX3KG X1 = A or V, X2 = S or I, X3 = V or E. Preferably, X1 = A or V, X2 = I and X3 = V or E. SEQ ID No:188 045 IgHV3-23-1 CDR3 DRGWGSDX1 where X1 = Y or C SEQ ID No:189 058 IgHV3-11-3 CDR1 DYYMX1 where X1 = Y or S SEQ ID No:190 058 IgHV3-11-3 CDR2 X1ISX2X3X4SYTX5YX6DSVKG where X1 = T or Y, X2 = D or S, X3 = D or S, X4 = G or S, X5 = Y or N, X6 = P or A SEQ ID No:191 062-064- IgHV4-30-2 * 1 CDR1 SGGX1SWS where X1 = Y or H
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068 SEQ ID No:192 062-064-068 IgHV4-302 * 1 CDR2 X1X2YHSGX3TYX4NPSLKS X1 = any amino acid, preferably C, Y, S or A, X2 = I or L, X3 = S or N, X4 = Y or D SEQ ID No:193 062-064-068 IgHV4-30-2 * 1 CDR3 SSYDX1LTD where X1 = F or I SEQ ID No:194 069-181 IgHV1-18-1 CDR1 X1YGIS where X1 = Y or N SEQ ID No:195 069-181 IgHV1-18-1 CDR2 WISX1YNGX2TNYAQKLQG where X1 = A or T, X2 = N or Y. Preferably X1 = A or T and X2 = Y SEQ ID No:196 069-181 IgHV1-18-1 CDR3 DLRGTX1YFDY where X1 = A or N SEQ ID No:197 098 IgHV1-18-1 CDR1 X1X2GIS where X1 = N or S,X2 = F or Y SEQ ID No:198 098 IgHV1-18-1 CDR2 WISAX1NGX2TX3YX4QKX5QG where X1 = F or Y, X2 = H or N, X3 = D or N, X4 = S or A, X5 = V or L SEQ ID No:199 101 IgHV1-18-1 CDR1 X1X2GIX3 where X1 = R or S, X2 = H or Y, X3 = T or S SEQ ID No:200 101 IgHV1-18-1 CDR2 WISAX1NGNTNYAQKX2QX3 where X1 = D or Y, X2 = F or L, X3 = D or G SEQ ID No:201 101 IgHV1-18-1 CDR3 VX1RYFD W LLX 2YFDY where X1 = F or L,X2 = P or in aa SEQ ID No:202 005-006 IGKV1D-16 * 01 CDR3 QQYNSX1PX2T X1 = Y or F, X2 = P or W. Preferably, X1 = Y or F and X2 = P SEQ ID No:203 008-035 IGKV1-13 * 02 CDR2 X1ASSLES where X1 = D, V or A SEQ ID No:204 008-035 IGKV1-13 * 02 CDR3 QQFNSYPLX1T where X1 = R, I, L,W or MY SEQ ID No:205 022 IGKV1-12 * 01 CDR3 QX1X2X3SFX4WT where X1 = Q or E, X2 = A or T, X3 = N or S; X4 = P or T SEQ ID No:206 024 IGKV1-12 * 01 CDR3 QQANSFPX1T where X1 = I or aa SEQ ID No:207 058 IGKV1-13 * 02 CDR3 QQFX1SYPX2IT where X1 = T or N,X2 = Q or in aa SEQ ID No:208 062-064-068 IGKV1-12 * 01 CDR3 QQANX1FPIT where X1 = G or S
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SEQ ID No:209 069-181 IGKV1-12 * 01 CDR1 RASQGISX1WLA where X1 = Y or N SEQ ID No:210 069-181 IGKV1-12 * 01 CDR2 AASSLX1S where X1 = Q or L SEQ ID No:211 098 IGKV1D-16 * 01 CDR3 X1QYX2SYPWT where X1 = H or Q,X2 = K or N SEQ ID No:212 101 IGKV3-20 * 01 CDR2 GX1X2SRAT where X1 = V or A,X2 = F or S
Example 12: Purification of antibodies [00252] Culture supernatant was filtered over 0.2 pm closed-end filters and loaded onto 5 ml MabSelect SuRe columns (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH , pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM NaH ₂ PO4, 140 mM NaCl, pH 7.4 (B. Braun). Alternatively, subsequent to purification, the eluate was loaded onto a HiPrep Desalting column and the antibody was exchanged for 12.6 mM NaH2PO4 buffer, 140 mM NaCl, pH 7.4 (B. Braun). After dialysis or buffer exchange, the samples were sterilely filtered over 0.2 pm closed-end filters. Purity was determined by SDS-PAGE and the concentration was measured by nephelometry and absorbance at 280n m. Purified antibodies were stored at 4 ° C. Mass spectrometry was performed to identify the molecular mass of the heavy and light chains of the antibody expressed by the hybridomas as described in Example 10.
Example 13: Binding of anti-c-Met clones to tumor cells expressing membrane bound c-Met measured by analysis using FACS [00253] The binding of anti-c-Met antibodies and their monovalent forms (also called here "UniBody molecules", see Example 5) in A431 cells expressing membrane-bound c-Met (acquired from ATCC, CRL-1555) was tested using flow cytometry (FACS Canto II, BD Biosciences). Qifi analysis (Dako, Glostrup, Denmark) revealed that A431 cells express an average of 30,000 copies of protein c
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Met per cell. Binding of anti-c-Met antibodies and UniBody molecules was detected using a goat anti-human IgG antibody conjugated to Phycoerythrin (Jackson). IgG1-5D5 was used as a positive control antibody, and HuMab-KLH was used as an isotype control antibody. EC50 values were determined using non-linear regression (sigmoid dose response curve with variable inclination) using the GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).
[00254] Figure 3 shows that all tested anti-c-Met antibodies and UniBody molecules bound to c-Met expressed on A431 cells in a dose-dependent manner. The EC50 values for binding varied between 0.28 nM and 1.92 nM for IgG and 0.52 nM and 13.89 nM for UniBody molecules. Interestingly, IgG1-024 antibody demonstrated high levels of unsaturated binding in A431 cells, which was not seen when binding in HT-29 cells (acquired from ATCC, HTB-38 ™) was tested (data not shown). For antibodies 022, 024, 062, 064, 069, 098, 101 and 181, no EC50 values or EC50 values less than 2 times decreased between IgG1's and UniBody molecules from identical clones were observed. Maximum binding levels also remained unchanged between IgG1's and UniBody molecules. For antibodies 005, 006, 008, 035, 045 and 058, on the other hand, a greater than 2-fold decrease in the EC50 value as well as a decrease in the maximum binding level were observed when comparing IgG1 with its counterpart UniBody. This was most likely due to the lower dissociations (Kd) of these antibodies (see Example 14).
Example 14: Octet affinity rating assay [00255] Antibody binding in cMetECDHis was analyzed using Bio-layer Interferometry (BLI) technology in the Octet System (Fortebio, Menlo Park, USA). Anti-human IgG (Fpecific) biosensors were used to capture anti-c-Met antibodies according to the
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97/121 procedure recommended by the manufacturer. cMetECDHis derived from HEK293 cells was loaded on top of immobilized anti-c-Met antibodies by positioning the loaded biosensor inside a well containing 10 µg / mL of cMetECDHis diluted 10 times with kinetic buffer (Fortebio). The difference in light reflection (Δλ, nm) from the biosensor surface due to the cMetECDHis binding was measured in real time for approximately 10 minutes and was used by the Octet software (V4.0, Fortebio) to calculate the association constant (ka [1 / M xs]). Then, the loaded biosensor was placed inside a well containing only kinetic buffer (10 times diluted in PBS) to determine the dissociation constant (kd [1 / s]). Kinetic analysis was performed to determine the affinity (Kd [M]) using the 1: 1 model (langmuir). As a positive control, 0.2 pg / ml of 5D5 IgG1 produced in HEK293 cells was used.
[00256] Table 5 shows that all anti-c-Met antibodies bound to cMetECDHis with nanomolar affinities within the 0.6-13.9 nM range.
Table 5: Kinetic constants (ka, kd and Kd) of antibodies for binding in cMetECDHis
Clone K [1 / Ms] kd [1 / s] Kd [M] 5D5 2.14E + 05 1.25E-03 5.86E-09 005 3.18E + 05 2.52E-03 7.92E-09 006 4.25E + 05 4.20E-03 9.89E-09 008 3.08E + 05 1.57E-03 5.12E-09 022 2.36E + 05 2.51E-04 1.06E-09 024 1.45E + 05 2.28E-04 1.57E-09 035 2.64E + 05 3.68E-03 1.39E-08 045 7.21E + 05 2.07E-03 2.87E-09 058 4.64E + 05 1.25E-03 2.70E-09 061 2.56E + 05 1.53E-04 5.96E-10 062 2.73E + 05 3.19E-04 1.17E-09 064 2.84E + 05 3.24E-04 1.14E-09 068 3.21E + 05 1.35E-03 4.21E-09 069 2.12E + 05 2.67E-04 1.26E-09 096 1.96E + 05 5.00E-04 2.55E-09 098 1.64E + 05 2.97E-04 1.82E-09
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101 1.69E + 05 2.14E-04 1.27E-09 181 2.37E + 05 5.31E-04 2.23E-09
[00257] Except for 5D5, each sample was measured once.
Example 15: Binding of anti-c-Met antibodies to membrane-bound c-Met expressed on Rhesus monkey epithelial cells measured by analysis using FACS [00258] To determine cross-reactivity with Rhesus monkey c-Met, binding of anti-c-Met antibodies on c-Met positive Rhesus monkey epithelial cells (4MBr-5 acquired from ATCC) was tested by flow cytometry (FACS Canto II, BD Biosciences). A goat anti-human IgG antibody conjugated to Phycoerythrin (Jackson) was used as a secondary conjugate. HuMab-KLH was used as an isotype control antibody.
[00259] Figure 4 demonstrates that all tested anti-c-Met antibodies cross-react with Rhesus monkey c-Met. At both concentrations tested (0.5 pg / ml and 10 pg / ml) anti-c-Met antibodies were able to specifically bind to Rhesus monkey c-Met. For all antibodies, the signal was at least 5 times higher than that for the HuMab-KLH isotype control antibody. Interestingly, P1016035 demonstrated much higher top fluorescence levels (MFI ~ 200,000) compared to other c-Met specific antibodies. This difference was not seen in cell lines expressing human cMet receptor.
Example 16: Blocking HGF binding in the extracellular domain of c-Met determined with the Enzyme-Linked Immunosorbent Assay (ELISA) [00260] An ELISA was performed to analyze for anti-c-Met antibodies that could block the growth factor binding of hepatocyte (HGF) at c-Met receptor. Therefore, c-Met coated extracellular domain was incubated with an unlabeled anti-c-Met antibody and HGF
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99/121 fluorescently marked. Non-blocking antibodies do not compete with HGF labeled for c-Met binding, resulting in maximum fluorescence signal. Blocking antibodies compete with HGF labeled for c-Met binding, resulting in a decreased fluorescence signal.
[00261] HGF (ProSpec Tany, Rehovot, Israel) was fluorescently marked by conjugation with Európio3 + (PerkinElmer, Turku, Finland). ELISA wells were coated overnight at 4 ° C with 0.5 pg / ml recombinant human c-Met extracellular domain (R&D systems, Minneapolis, USA) diluted in PBS. Next, the ELISA wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [SigmaAldrich, Zwijndrecht, Netherlands]) and blocked for one hour at room temperature (RT) with PBST supplemented with 2% (v / v) of chicken serum (Gibco, Paisley, Scotland). After washing with PBST, the ELISA wells were incubated for one hour at RT protected from light with a mixture of 50 μl of serially diluted anti-c-Met antibody (0.128-10.000 ng / mL in 5-fold dilutions) and 50 pL of HGF conjugated to Europio3 + 0.44 pg / mL in PBST. Then, HGF conjugated with unalloyed Europium ³⁺ was removed by washing with PBST and HGF conjugated with bound Eurium ³⁺ was incubated for 30 minutes in RT in the dark with Delfia Enhancement Solution (PerkinElmer) to increase the fluorescent signal. The fluorescence intensity at 615 nm was measured using the EnVision 2101 Multilabel reader (PerkinElmer) applying the following configurations: Lance / Delfia dual mirror, 615 emission filter, 340 nm excitation filter, 400 ps delay time, 400 ps window , 100 flashes, 2000 ps per cycle and bidirectional reading from row to row. To determine the IC50 values, the binding curves were analyzed with non-linear regression (sigmoid dose-response curve with variable slope, top values restricted to a shared value for all data sets) using the GraphPad Prism V4 software. 03 (GraphPad Software, San Diego, CA, USA).
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100/121 [00262] Figure 5 shows representative examples of HGF binding inhibition curves of anti-c-Met antibodies for binding in the extracellular domain of recombinant human c-Met. 5D5 was used as a positive control antibody. All anti-c-Met antibodies in the experiment shown were able to compete with HGF conjugated to Europe ³⁺ for binding to recombinant c-Met. IC50 values ranged between 0.0011 pg / mL and 0.0794 pg / mL. Without the addition of HGF conjugated to Europeium ³⁺ , approximately ~ 600 relative fluorescent units (RFU) were detected, indicating the signal when maximum inhibition was achieved. When binding in HGF conjugated to Europeus ³⁺ was not inhibited, approximately ~ 66,000 RFU were detected. Antibodies 005, 006, 058, 101 and the positive control antibody 5D5 were able to inhibit 84.5-92.1% of HGF binding at the c-Met receptor. All other antibodies were able to inhibit at least 55% HGF binding in c-Met. Since HGF can bind to the c-Met receptor in both the SEMA and Ig regions, some antibodies can inhibit just one of these interactions. To determine which interaction was inhibited, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay based on cMetSEMAHis was performed.
Example 17: Competition of anti-c-Met antibodies for binding in soluble cMetECDHis measured with sandwich ELISA [00263] First, the optimal coating concentrations of the tested anti-c-Met antibodies and the optimal concentration of cMetECDHis were determined. Therefore, ELISA wells were coated overnight at 4 ° C with anti-c-Met HuMabs serially diluted in PBS (8 pg / mL in double dilutions). Then, the ELISA wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [SigmaAldrich, Zwijndrecht, Netherlands]) and blocked for one hour at room temperature (RT) with PBSTC (PBST supplemented with 2% [v / v]
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101/121 of chicken serum [Gibco, Paisley, Scotland]). Subsequently, the ELISA wells were washed with PBST and incubated for one hour in RT with biotinylated cMetECDHis serially diluted with PBSTC (1 pg / mL in double dilutions). Unbound biotinylated cMetECDHis was removed by washing with PBST, and bound biotinylated cMetECDHis was incubated for one hour in RT with 0.1 pg / ml streptavidin-poly-HRP (Sanquin, Amsterdam, Netherlands) diluted with PBST. After washing, the reaction was visualized during a 15 minute incubation with 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS tablet in 50 mL of ABTS buffer [Roche Diagnostics , Almere, Netherlands]) at RT protected from light, staining was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, Netherlands). Fluorescence at 405 nm was measured on a plate reader microtiter (Biotek Instruments, Winooski, USA) The conditions that resulted in sub-optimal binding (approximately 80%) of each antibody were determined and used to follow cross-blocking experiments.
[00264] ELISA wells were coated with anti-cMet antibody in a sub-optimal dose as described above. After blocking the ELISA wells, they were incubated with the predetermined concentration of biotinylated cMetECDHis in the presence of an excess of anti-c-Met antibody. The reaction was developed as described above. Residual binding was expressed as a percentage relative to the binding observed in the absence of competing antibody.
[00265] Table 6: When added as competitors, all anti-c-Met antibodies were able to compete for binding with their immobilized counterparts. 022, 058 and 5D5, when added as competing antibodies, competed with antibodies 005 and 006. However, the reverse reaction revealed only partial competition for antibodies 005 and 006. These differences can be explained by the lower affinities of
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102/121 antibodies 005 and 006 by biotinylated cMetECDHis. Antibody 5D5, when added as a competitor antibody, also demonstrated partial competition with antibodies 008 and 045, while no competition or minimal competition was observed in the reverse reaction. In addition, antibodies 024, 062, 064, 068 and 181, when added as competing antibodies, demonstrated partial competition with antibody 101, while the reverse reaction demonstrated complete inhibition of binding in cMetECDHis. Values higher than 100% can be explained by the effects of avidity and the formation of antibody-cMetECDHis complexes containing two non-competing antibodies.
[00266] Antibodies 024, 062, 064, 068, 069, 098, 101 and 181 compete with each other for connection in cMetECDHis. Antibodies 005, 006, 022 and 058 were considered to belong to a cross-blocking group, a group that is characterized by complete competition with 005, 006, 022, 058 and 5D5. However, antibody 5D5 was the only antibody that was also able to compete for binding with antibody 045. Another group of antibodies that compete for binding in cMetECDHis is formed by 008, 035 and G11-HZ.
Table 6: Competition of anti-c-Met antibodies for binding to biotinylated cMetECDHisCompetitive antibody Immobilized antibody 005 006 008 022 024 035 045 058 005 7.7 ± 1.1 18.2 ± 3.6 81.9 ± 3.1 4.9 ± 1.3 113.5 ± 5.0 84.9 116.9 3.6 ± 0.2 ± 7.0 ± 0.1 006 11.3 ± 0.9 14.6 ± 0.7 58.8 ± 2.2 4.6 ± 0.3 113.3 ± 1.0 67.5 114.5 3.6 ± 4.2 ± 3.5 ± 0.3 008 63.9 ± 3.1 47.3 ± 1.2 5.4 ± 0.3 82.1 103.2 ± 0.4 32.9 100.4 40.8 ± 3.0± 1.0 ± 3.8 ± 0.8 022 37.9 ± 3.9 60.5 ± 4.0 94.1 ± 3.5 3.8 ± 1.2 99.4 ± 4.8 92.4 95.7 5.8 ± 0.4 ± 3.5 ± 0.0 024 98.4 101.4 * 104.2 * 100.2 * 5.4 ± 0.5 108.1 * 98.1 * 102.8 *± 10.4 ± 16.7 ± 12.7 ± 9.0± 5.8 ± 11.9 ± 12.8 035 36.7 ± 1.0 33.0 7.2 ± 1.7 54.6 121.4 ± 27.8 10.6 125.0 18.5 ± 17.6± 6.5± 0.3 ± 16.8 ± 2.5 045 111.4 110.6 98.5 ± 3.1 105.3 102.4 ± 5.6 105.4 21.3 115.3 *± 1.5 ± 3.5± 2.5± 5.5 ± 0.1 ± 6.5 058 31.4 ± 3.6 43.6 ± 2.1 90.2 ± 2.5 6.8 ± 0.3 109.0 ± 4.1 90.1 111.7 4.0 ± 5.4 ± 4.9 ± 0.2 062 95.8 ± 5.1 95.2 ± 6.8 97.4 ± 5.3 94.6 7.3 ± 2.9 90.6 97.0 94.4 ± 4.0± 11.5 ± 3.0 ± 4.3 064 90.4 ± 1.9 90.1 * 94.6 * 94.2 7.5 ± 2.5 83.5 95.0 95.5
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± 1.4 ± 0.5 ± 3.6± 12.2 ± 4.9 ± 0.6 068 101.1 98.5 ± 6.7 101.7 99.6 4.7 ± 2.3 88.6 100.4 101.5± 7.6± 5.5 ± 4.0± 12.7 ± 9.0 ± 5.1 069 102.3 100.3 102.1 97.8 6.6 ± 4.1 91.7 99.8 100.6± 11.2 ± 12.3 ± 12.8 ± 12.5± 27.3 ± 14.4 ± 14.1 098 99.6 ± 6.3 97.9 ± 6.7 99.8 ± 4.2 95.8 12.9 ± 4.2 89.4 96.7 98.6 ± 5.4± 20.6 ± 3.7 ± 2.9 101 91.5 ± 7.2 89.7 ± 7.9 94.0 ± 6.3 90.7 40.5 ± 5.4 96.7 94.7 93.1 ± 5.3± 1.9 ± 5.1 ± 5.2 181 95.9 ± 7.8 93.7 ± 8.4 98.7 ± 5.8 92.5 4.3 ± 1.9 96.0 96.8 98.9 ± 7.4± 9.6 ± 6.7 ± 9.8 5D5 42.3 58.8 90.2 ± 9.9 12.4 94.2 ± 9.7 98.1 83.9 6.6± 14.7 ± 19.4± 4.7 ± 13.4 ± 3.2 G11-HZ 50.5 ± 7.6 47.7 ± 2.9 33.3 ± 0.2 54.3 98.8 ± 5.6 32.8 72.0 27.6 ± 3.7± 4.0 ± 9.9 ± 4.3
- competition> 100%
- 74% competition
- 24% competition
Competitive antibody Immobilized antibody 062 064 068 069 098 101 181 5D5 G11-HZ 005 117.7 118.2 128.7 124.0 110.4 103.2 131.0 2.9 76.8± 10.7 ± 7.8 ± 9.5 ± 8.0 ± 7.6 ± 5.0 ± 7.7 ± 0.1 ± 4.4 006 118.8 122.2 128.6 124.5 110.6 105.9 123.5 3.1 54.0± 8.4 ± 5.3 ± 6.5 ± 1.0 ± 2.3 ± 4.1 ± 6.1 ± 0.0 ± 35.1 008 100.5 107.1 112.2 104.1 106.6 101.0 111.3 32.4 2.7± 2.5 ± 6.2 ± 5.1 ± 4.4 ± 2.6 ± 2.5 ± 1.3 ± 0.8 ± 0.2 022 99.4 101.9 104.1 99.6 104.8 103.6 107.1 4.2 85.9± 2.0 ± 3.2 ± 3.3 ± 6.0 ± 4.0 ± 5.1 ± 5.2 ± 2.1 ± 8.3 024 2.3 2.3 12.0 2.9 10.4 4.8 7.1 95.5 98.2 *± 0.6 ± 0.6 ± 5.5 ± 0.5 ± 4.2 ± 1.0 ± 2.8 * ± 1.3 ± 1.1035 119.6 131.7 175.1 150.9 126.2 113.0 159.1 25.5 7.8± 11.2 ± 20.0 ± 30.2 ± 24.9 ± 19.9 ± 4.6 ± 12.9 ± 9.9 ± 3.2 045 103.1 103.7 113.1 97.0 76.4 101.5 99.4 27.8 99.3± 3.5 ± 5.7 ± 1.4 ± 5.2 ± 11.7 ± 5.1 ± 3.8 ± 3.9 ± 5.3 058 109.1 108.8 118.8 112.6 111.8 104.4 121.3 2.8 81.5± 4.6 ± 4.4 ± 4.2 ± 4.0 ± 6.2 ± 0.8 ± 3.1 ± 0.4 ± 8.6 062 2.4 2.2 14.2 2.9 13.2 7.8 9.4 97.7 101.3± 0.5 ± 0.2 ± 1.8 ± 0.1 ± 0.9 ± 1.1 ± 1.6 ± 8.5 ± 0.9 064 2.2 2.0 13.0 2.7 14.7 7.6 10.1 94.9 102.0± 0.6 ± 0.2 ± 0.9 ± 0.2 ± 1.2 ± 0.8 ± 3.0 * ± 10.5 ± 4.6068 2.0 2.0 6.6 2.4 8.2 4.8 5.2 94.8 110.3± 0.3 ± 0.3 ± 0.7 ± 0.4 ± 1.3 ± 0.7 ± 0.6 ± 2.7 ± 6.6 069 2.2 2.3 10.1 2.4 12.5 3.9 6.3 99.4 110.4± 0.4 ± 0.5 ± 2.6 ± 0.7 ± 3.1 ± 0.5 ± 1.0 ± 16,2 ± 13.2 098 8.8 9.3 18.0 3.4 2.6 4.0 12.0 94.9 99.6± 0.6 ± 1.3 ± 2.5 ± 0.6 ± 0.4 ± 0.6 ± 2.1 ± 1.2 ± 1.2 101 36.9 37.4 45.9 9.5 9.7 3.7 41.9 97.2 98.3± 3.3 ± 3.7 ± 4.3 ± 1.2 ± 1.5 ± 2.4 ± 0.8 ± 4.6 ± 2.1
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181 2.0 2.1 6.5 2.2 5.1 2.4 3.6 94.2 98.7± 0.2 ± 0.3 ± 1.1 ± 0.3 ± 1.1 ± 0.2 ± 0.2 ± 4.5 ± 6.7 5D5 97.6 97.1 97.8 99.6 97.6 97.9 103.4 4.1 97.3± 8.1 ± 12.7 ± 6.6 ± 3.9 ± 4.9 ± 10.6 ± 4.3 ± 1.5G11-HZ 95.3 99.2 102.6 95.0 96.2 90.1 101.1 29.1 2.6± 3.1 ± 0.6 ± 1.3 ± 8.4 ± 11.8 ± 6.8 ± 5.2 ± 9.2 ± 0.4
[00267] Data shown are percentages of binding inhibition ± the standard deviation of 3 independent experiments. For antibodies 035, 5D5 and G11-HZ the cross-block ELISA was performed only twice. In addition, numerous competition reactions (*) have resulted in Optical Density values greater than 5.0, which are above the detection limit of the ELISA reader. These results were disregarded from the analysis resulting in measurements taken twice.
Example 18: HGF binding block in cMetSEMA567His8 determined by time resolved fluorescence resonance energy transfer (TR-FRET) [00268] HGF can bind to the c-Met receptor in both the SEMA domain and the IgG region . However, only HGF bound in the SEMA domain was found to be crucial for receptor activation. Therefore, the interaction of anti-c-Met antibodies with the SEMA domain of the c-Met receptor has been studied using TR-FRET technology. In order to carry out this assay based on homogeneous proximity, hepatocyte growth factor (HGF, ProSpec Tany, Rehovot, Israel) was combined with a fluorescent acceptor dye; AlexaFluor-647 (Invitrogen, Breda, Netherlands). cMetSEMA-567His8 was labeled with a fluorescent donor molecule against the histidine tag (Anti-6xhis Europium ³⁺ , PerkinElmer, Turku, Finland). Binding of HGF conjugated to AlexaFluor647 in cMetSEMA-567His8 labeled with Europium ³⁺ allows an energy transfer from the donor molecule (excitation at 340 nm) to the acceptor molecule (emission at 665 nm). The average fluorescent intensity at 665 nm was measured on the EnVision 2101 Multilabel reader (PerkinElmer). Competition of unlabeled anti-c-Met antibodies with HGF conjugated to
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AlexaFluor-647 was measured by a decrease in TR-FRET signal at 665 nm, because in the unbound state, the distance between the donor and acceptor fluorophores is too large for energy transfer to occur.
[00269] All dilutions were made in 0.5x Lance detection buffer (PerkinElmer) supplemented with 2.67% Stabilizer solution (PerkinElmer) and 0.03% (v / v) Tween-20 (Riedel de Haen, Seelze , Germany). 25 pL of cMetSEMA-567His8 were added in 25 pL of HGF conjugated to AlexaFluor-647, 25 pL of anti-6xhis Europium ³⁺ and 25 pL of unlabeled anti-c-Met antibody in a 96-well opti-white plate PerkinElmer). A final concentration of 2.93 pg / ml of cMetSEMA-567His8, 0.96 pg / ml of HGF conjugated to AlexaFluor647 and 0.4 pg / ml of anti-6xhis Europio ^{3+ was obtained} . A quadruple serial dilution of unlabeled anti-c-Met antibody ranging from 0.49 - 8,000 ng / mL was tested. After overnight incubation at 4 ° C in the dark, the average fluorescence intensity at 665 nm was measured using EnVision 2101 Multilabel reader applying the following configurations: Lance / Delfia dual mirror, emission filter 615-665 nm, excitation filter 320 nm, delay time 60 ps, window 100 ps, 100 flashes, 2000 ps per cycle and bidirectional reading from row to row. To determine the IC50 values, the binding curves were analyzed with non-linear regression (sigmoid dose-response curve with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).
[00270] Figure 6 shows the HGF binding inhibition curves of the various anti-c-Met antibodies for binding in cMetSEMA_567His8 tested with TR-FRET. Except for antibodies 008, 035 and 096, all antibodies were able to compete with HGF conjugated to AlexaFluor-647 for binding in cMetSEMA-567His8. Antibody 022 was able to inhibit ~ 80% of HGF binding, while antibodies 005, 006, 024, 045, 058, 061, 062, 064, 068, 069, 098, 101, 181 and the positive control antibody 5D5
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106/121 were able to inhibit> 90% HGF binding in cMetSEMA-567His8. IC 50 values ranging from 0.082-0.623 pg / mL were determined.
Table 7: IC50 values (pg / mL) and percentage of anti-c-Met antibody ligand inhibition for cMetSEMA567His8 binding determined with TR-FRET
mAb IC50 Inhibition% 005 0.16 92 006 0.16 92 008 ND 4 022 0.37 77 024 0.39 95 035 ND 19 045 0.17 92 058 0.15 99 061 0.49 96 062 0.58 97 064 0.07 97 068 0.26 96 069 0.54 97 096 ND 16 098 0.55 98 101 0.53 96 181 0.34 93 5D5 0.2 95
[00271] Data shown is average MFI from three independent experiments.
Example 19: KP4 Viability Assay [00272] Anti-c-Met antibodies were tested for their ability to inhibit the viability of KP4 cells (Riken BioResource Center Cell Bank, RCB1005). KP4 cells, which express high levels of both c-Met and HGF in an autocrine manner, were seeded in a 96-well tissue culture plate (Greiner bio-one, Frickenhausen, Germany) (10,000 cells / well) in free medium of serum (1 part of HAM's F12K [Cambrex, East Rutherford, New Jersey] and 1 part of DMEM [Cambrex]). Dilution of 66.7 nM anti-c-Met antibody in serum-free medium was prepared and added to the cells. After a 3-day incubation, the amount of viable cells was quantified with Alamar blue (BioSource International, San Francisco, US)
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107/121 according to the manufacturer's instruction. Fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard calibrations with Alamar blue. The Alamar blue signal from antibody-treated cells was plotted as a percent sign compared to untreated cells.
[00273] Figure 7 shows the percentage inhibition of viable KP4 cells after treatment with anti-c-Met antibody compared to untreated cells (0%). Clones within a rectangle are antibodies that cross-compete with each other as described in Example 17. Interestingly, antibodies 024, 062, 064, 068, 069, 098, 101 and 181, which belong to the same cross-block group, were all capable of inhibiting the viability of KP4 (18-46%), both as IgG1 and as a UniBody molecule. IgG1 molecules of antibodies 008, 061 and 096 were also able to inhibit the viability of KP4. In contrast, antibody 045 did not inhibit the viability of KP4 as IgG1 or as a UniBody molecule. For Uni-1016-045-TE this may be due to its low apparent affinity for membrane-bound c-Met, as measured by analysis using FACS (Example 13). IgG1 antibodies from clones 005, 006, 022 and 058 did not significantly inhibit KP4 viability, whereas Uni-1016-022-TE, Uni-1016-058-TE and IgG1-1016-058-wtFab inhibited 57% , 38% and 44% the viability of KP4, respectively. Uni-1016-005 and Uni1016-006 also cross-compete with clones 022 and 058 but did not significantly inhibit KP4 viability. This may be due to their low apparent affinities as measured by the analysis using FACS (Example 13). Interestingly, IgG4-1016-058 also demonstrated some inhibition of KP4 viability. This was not seen with IgG4-5D5.
[00274] Altogether the data indicate that for some cross-blocking groups, monovalent binding is required to inhibit KP4 viability, while for other cross-blocking groups both antibodies
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108/121 monovalent and bivalent can inhibit the viability of KP4.
Example 20: KP4 xenograft tumor model in SCID mice [00275] A KP4 xenograft tumor model in SCID mice was performed to determine the effectiveness of anti-Met HuMabs to inhibit in vivo growth. Eleven-week-old female SCID mice, strain CB-17 / IcrPrkdc-scid / CRL, were purchased from Charles River Laboratories Nederland (Maastricht, Netherlands) and kept under sterile conditions in top filter cages with food and water provided ad libitum. Microchips (PLEXX BV, Elst, Netherlands) were positioned for mouse identification. All experiments were approved by the Utrecht University Animal Ethics Committee.
[00276] On day 0, 10x10 ⁶ KP4 cells were inoculated subcutaneously in 200 pL of PBS on the right flank. The mice were examined at least twice a week for clinical signs of disease. The tumor size was determined at least once a week. Volumes (mm3) are calculated from caliper measurements (PLEXX) as 0.52 x (length) x (width) 2, starting on the day
16. On day 9, average tumor sizes were measured and the mice were divided into 8 groups of 7 mice each. Anti-c-Met antibodies (008, 058, 069 and 098) were injected intraperitoneally. G11HZ antibody was used as a positive control antibody, while 5D5 and isotype control antibodies were used as negative control antibodies. Mice received a loading dose of 400 pg / mouse followed weekly by a maintenance dose of 200 pg / mouse for 7 weeks.
[00277] Additionally, plasma samples, collected before administration of 1 ^a , 3 ^a and 5 ^a maintenance doses and when the mice were finished, the presence of human IgG was verified using beads
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109/121 latex in the BNII nephelometer (Dade Behring, Atterbury, United Kingdom). [00278] Figures 8 and 9 show that tumor growth of KP4 cells was inhibited by HuMabs 008, 069, 098 and positive control G11-HZ. Inhibition was compared to treatment with isotype control antibody. Tumor growth of KP4 cells was slowed but not completely inhibited by the control antibody G11-HZ. Clones 069 and 098 showed more potent inhibition compared to clones 008 and G11HZ. Antibodies 5D5 and 058 did not inhibit tumor growth. This was consistent with the in vitro data as described in Example 19. Taken together, these data indicate that for some cross-blocking groups bivalent binding antibodies can inhibit KP4 tumor growth.
Example 21: MKN45 xenograft tumor model [00279] A model of human MKN45 gastric adenocarcinoma xenograft in nude mice was used to determine the effectiveness of anti-c-Met HuMabs to inhibit tumor growth in vivo. [00280] MKN45 human gastric adenocarcinoma cells were cultured at 37 ° C and 5% CO2 in RPMI-1640 medium containing 100 units / ml of sodium penicillin, 100 pg / ml of streptomycin sulfate, 25 pg / ml of gentamicin, 20% fetal bovine serum, and 2 mM glutamine. Female nude mice aged seven to eight weeks (nu / nu, Harlan) (body weights ranging from 17.0 to 26.4 g at the beginning of the study) were used. The animals were fed water and food ad libitum. The mice were housed under conditions according to the recommendations of the “Guide for Care and Use of Laboratory Animals”. The animal use and care program was accredited by AAALAC. On day 0, 1x10 and ⁷ MKN45 cells were inoculated subcutaneously in 200 µl of 50% matrigel in PBS on the flank of each mouse. On day 7, the animals were divided into five groups (n = 10) with an average tumor volume of 80 to 120 mm ³ and
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110/121 treatment was started. Anti-c-Met antibodies (008, 058, 069) were injected into the tail vein (iv). G11-HZ antibody was used as a positive control antibody and an isotype control antibody was used as a negative control antibody. All mice received 40 mg / kg of antibody on day 7 and 20 mg / kg of antibody on days 14, 21, and 28.
[00281] Tumors were measured weekly using calipers up to an endpoint tumor volume of 700 mm3 or until the end of the study (day 62). Figures 10 and 11 show that tumor growth of MKN45 cells was significantly retarded by antibodies 008, 058, 069 and by the control antibody G11-HZ compared to treatment with the isotype control antibody.
Example 22: Decreasing residual agonistic activity of anti-c-Met IgG1 antibodies by reducing conformational flexibility [00282] The natural c-Met ligand, HGF, is a functional dimer that induces the dimerization of two c-Met molecules. Subsequent intracellular phosphorylation of the intracellular domain of c-Met results in the activation of several signaling routes that are involved in cell proliferation, invasion and survival. Bivalent antibodies against c-Met mostly show comparable effects such as HGF on cell death, especially when the antibody-binding epitopes are located close to or in the SEMA domain of c-Met.
[00283] To minimize the potential residual agonistic activity of bivalent IgG1 antibodies, a strategy was used to reduce conformational flexibility. In an IgG1, there is a high degree of freedom for the Fab arms to move in relation to the Fc domain. Major conformational changes are the result of joint flexibility, which allows for a wide range of Fab-Fc angles (Ollmann Saphire, E., R.L. Stanfield, M.D.M. Crispin, P.W.H.I. Parren, P.M. Rudd, R.A. Dwek, D.R.
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Burton and I.A. Wilson. 2002. “Contrasting IgG structures reveal extreme asymmetry and flexibility”. J. Mol. Biol. 319: 9-18). One way to reduce the flexibility of the Fab arm in immunoglobulins is to avoid the formation of disulfide bonds between the light chain and the heavy chain through genetic modification. In a natural IgG1 antibody the light chain is covalently connected to the heavy chain via a disulfide bond, connecting the C-terminal cysteine of the light chain to the cysteine at position 220 (EU number C220) at the Fc joint of the heavy chain. Whether by mutating amino acid C220 to serine or any other natural amino acids, removing C220, removing the entire joint, or replacing the IgG1 joint with an IgG3 joint, a molecule is formed in which the light chains are connected via its C-terminal cysteines, analogous to the situation found in the human IgA2m isotype (1). This results in a reduced flexibility of the Fabs relative to Fc and consequently reduced cross-linking capacity, as shown in comparative studies with IgG1 and IgA2m (1) formats of an agonistic anti-c-Met (5D5) antibody in a viability assay of KP4 (Figure 12). [00284] Another strategy to reduce the flexibility of an IgG1 molecule is to replace the IgG1 joint with the IgG2 joint or with the IgG2-like joint. (Dangl et al. EMBO J. 1988; 7: 1989-94). This joint region has two distinct properties from that of IgG1, which considerably make the molecules less flexible. First, compared to the IgG1 joint, the IgG2 joint is 3 amino acids shorter. Second, the IgG2 joint contains an additional cysteine, so three will be formed instead of two heavy interchain disulfide bridges. Alternatively, a variant of the IgG1 joint that looks like the IgG2 joint can be introduced. This mutant (ΤΗ7Δ6-9) (WO2010063746) contains a T223C mutation and two deletions (K222 and T225) in order to create a shorter joint with a
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Example 23: Generation of IgG1 molecules with reduced flexibility (stiffened IgG1 molecules)
Cloning and expression [00285] Mutant IgG1 antibodies were designed and cloned using standard molecular biology techniques. An overview of the sequences of all the hinge region mutations generated is shown in Table 8 below.
Table 8: Joint amino acid sequence of mutant IgG1 antibodies. Deletions are marked with '-' and mutations are underlined.
IgG1 WT EPKSCDKTHTCPPCP
Joint IgG1-IgG2 ERKCCVE --- CPPCP
IgG1 AC220 EPKS-DKTHTCPPCP
IgG1 AC22S EPKSSDKTHTCPPCP
IgG1 TH7A6-9 EPKSCD-CH-CPPCP
IgG1 with deleted joint (Uni-IgG1) -------------------------- IgG1 joint - ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP
IgG3 [00286] For the expression of the resulting stiffened IgG1 antibodies in mammalian cells, the HC constant region of IgG1, containing mutations in the articulation region (see Table 8 above), was synthesized as a codon-optimized construct in vector of mammalian expression pcDNA3.3 (Invitrogen). A separate vector was constructed by inserting the codon-optimized constant region of the human kappa light chain region into pcDIMA3.3. VH and VL regions of clone 069 and control antibody 5D5 were inserted into the HC constant plasmid and Kappa light chain plasmid respectively resulting in vectors for the expression of the light and heavy (mutated) chains of the specific antibodies. Cotransfection of the heavy and light chain vectors of a specific antibody in HEK-293F cells (Invitrogen), resulted in the transient production of mutant antibodies. Purification of the antibodies was performed using Protein A affinity column chromatography (as described in Example 11).
Biochemical characterization
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Transient expression [00287] All mutants were expressed at sufficient levels and did not show aberrant multimer formation as determined by MS (purity> 99%) and SDS-PAGE.
[00288] The results of SDS-PAGE are shown in Figure 13. In mutants C220 (C220S and AC220) and variants of IgG1 with deleted joint (variants of IgG1 with deleted joint are also called UniBody-IgG1 or Uni-IgG1 ) light chain matching was observed, visible as a protein band of about 50 kD in unreduced SDS-PAGE analysis. The variant with an IgG3 joint also showed light chain pairing, while the variant with an IgG2 joint and the IgG1 mutant ΤΗ7Α6-9 showed normal light-heavy chain pairing.
Example 24: C-Met binding properties of the mutants [00289] c-Met binding properties of the mutants were tested in an ELISA. ELISA plate wells were coated overnight at 4 ° C with rhHGF R / Fc Chimera (R&D Systems; Cat.358MT / CF) in PBS (1 pg / ml). Then, the wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, Netherlands)] and blocked for one hour at room temperature (RT) with PBSTC (PBST supplemented with 2 % [v / v] of chicken serum [Gibco, Paisley, Scotland]). Subsequently, the wells were washed with PBST and incubated for one hour at RT with anti-cMet antibodies and variants serially diluted in PBSTC (10 pg / mL in quadruple dilutions). Unbound antibody was removed by washing with PBST, and antibody bound to the coating was detected by incubation for one hour at RT with goat anti-human IgG F (ab ') 2-HRP antibody diluted in PBST (Jackson, catalog number 109-035-097). After washing, the reaction was
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114/121 visualized by a 15 min incubation with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS: dilute an ABTS tablet in 50 mL of ABTS buffer [Roche Diagnostics, Almere, Countries -Basses]) in RT protected from light. Staining was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, Netherlands). Fluorescence at 405 nm was measured in a microtiter plate reader (Biotek Instruments, Winooski, USA). All mutants bound with comparable apparent affinities (EC50) in c-Met (Figure 14). Table 10 shows the EC50 values of the mutants obtained in this experiment.
Table 9: EC50 values as determined by ELISAIgG1-1016-069 IgG1-1016069-AC220 IgG1-1016069C220S IgG11016-069 IgG2 articulation IgG21016069 IgG1-1016- 069ΤΗ7Α6- 9 Uni1016069- TE IgG1-1016-069 IgG1-1016069articulationIgG3 Uni-IgG1-1016069 EC50 (ng / mL) 49.5 18.87 15.56 23.03 29.61 18.81 30.08 45.43 14.18 15.39
Example 25: Reduced agonistic effect of stiffened anti-c-Met IgG1 antibodies
Receptor phosphorylation [00290] To determine the agonistic properties of the stiffened antibodies, the antibody was made on the phosphorylation of c-Met. With the dimerization of two adjacent c-Met receptors either by natural ligand HGF or by mainly bivalent antibodies, three tyrosine residues (positions 1230, 1234 and 1235) in the intracellular domain of c-Met are cross-phosphorylated, which is followed by phosphorylation subsequent use of several other amino acids in the intracellular domain and by the activation of numerous signaling cascades. The dimerization and activation of c-Met can therefore be monitored by using specific antibodies to the phosphorylated receptor at these positions, and thus used as a reading for the potential agonism of anti-c-Met antibodies.
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115/121 [00291] A549, CCL-185 cells obtained from ATCC, were grown in DMEM medium containing serum until 70% confluence was reached. After trypsinization and washing the cells were plated on a 6-well culture plate at 1 * 10 ⁶ cells / well in culture medium containing serum. After overnight incubation, the cells were treated with either HGF (R&D systems; catalog number 294-HG) (50 ng / mL) or the antibody panel (30 pg / mL) and incubated for 15 minutes at 37 ° C . The cells were then washed twice with ice-cold PBS and lysed with lysis buffer (Cell Signaling; catalog number 9803) supplemented with a cocktail of protease inhibitors (Roche; catalog number 11836170001) and the samples were stored at -80 ° Ç. Receptor activation was determined by measuring phosphorylation by means of Western blot using specific antibodies to phospho-c-Met. The proteins in the cell lysate were separated on a 4-12% SDS-PAGE gel and transferred to a nitro-cellulose membrane which was subsequently stained with phosphorylated c-Met specific antibody (Y1234 / 1235) (Cell Signaling, number of catalog 3129). To control the loading of the gel, antibodies against c-Met and total beta-actin were used. Western blots results are shown in Figure 15.
[00292] Cells and controls in tissue culture medium treated with the monovalent 5D5 antibody UniBody format did not show receptor phosphorylation. In contrast, Western blot analysis of cells treated with the HGF positive control or IgG11016-058 agonist antibody showed an evident band at the expected time. IgG11016-069 antibody showed low, but detectable, phosphorylation of the receptor indicating that some cross-linking of the receptor occurs. However, variants that were designed to reduce the flexibility of the antibody molecule showed minimal receptor activation, below a level comparable to the levels detected in cells treated with Uni-5D5-TE monovalent control.
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116/121 (Figure 15).
Effect of anti-c-Met antibodies on NCIH441 proliferation in vitro [00293] The potential proliferative agonistic activity of anti-c-Met antibodies was tested using lung adenocarcinoma cell line NCI-H441 (ATCC, HTB-174 ™), which expresses high levels of c-Met, but does not produce its HGF ligand. NCI-H441 cells were seeded in a 96-well tissue culture plate (Greiner bio-one, Frickenhausen, Germany) (5,000 cells / well) in RPMI (Lonza) without serum. Dilutions of anti-c-Met antibody (66.7 nM) were prepared in RPMI without serum and added to the cells. After 7 days incubation at 37 ° C / 5% CO2, the quantity of viable cells was quantified with Alamar blue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with calibrations with standard Alamar blue.
[00294] As shown in Figure 17, the proliferation of NCIH441 cells was strongly induced by the agonistic control mAbs IgG1-058 and IgG1-5D5. IgG1-1016-069 antibody also showed some agonistic effect compared to cells treated with isotype control. The agonistic activity of IgG1-1016-069 could be completely removed by the introduction of the C220 C220S and -del mutants, and partially by the variants with the ΤΗ7Δ6-9 and IgG2 articulation or the main structure of IgG2. Control samples treated with isotype control and the monovalent version of 5D5 (Uni-5D5-TE) did not induce cell growth.
KP4 viability assay [00295] The ability to inhibit HGF-dependent cells was also determined for anti-c-Met antibody mutants in a KP4 viability assay (see Example 19 for experimental procedures). The
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117/121 results are shown in Figure 17. The efficacy of the IgG1-1016-069-based mutants was either completely retained or was slightly better in the C220 mutants. Notably, mutation of C220 in the 5D5 agonistic antibody resulted in a marked reduction in KP4 viability. No agonistic effects of antibodies 058 and 5D5 in IgG1 format were observed due to the high expression of HGF by KP4 (autocrine loop of HGF).
Inframodulation [00296] C-Met inframodulation induced by antagonistic antibodies represents a mechanism of action of therapeutic anti-c-Met antibodies. Consequently, in one embodiment, antibodies with reduced agonistic properties, but with retained ability to induce c-Met inframodulation, are desirable. To determine potential inframodulation of antibodies, A549 cells (CCL-185 obtained from ATCC) were seeded in 6-well tissue culture plates (500,000 cells / well) in cell culture medium containing serum and grown overnight at 37 ° C. ° C. The following morning, anti-cMet antibodies were added at a final concentration of 10 pg / mL and the plate was incubated for an additional 2 hours at 37 ° C. After washing with PBS, cells were lysed by incubation for 30 min at room temperature with 250 pL of Lysis buffer (Cell Signaling, Danvers, USA). Total protein levels were quantified using protein assay bicinconinic acid (BCA) reagent (Pierce) following the manufacturer's protocol. Levels of c-Met protein in cell lysates were quantified using a c-Met specific sandwich ELISA. For this purpose, ELISA plate wells were coated overnight at 4 ° C with goat anti-human c-Met antibody directed against the extracellular domain of c-Met (R&D systems), diluted with PBS (1 pg / mL). Then, the wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, Netherlands]) and blocked for one hour at RT with PBSTC (PBST
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118/121 supplemented with 2% [v / v] chicken serum [Gibco, Paisley, Scotland]). Undiluted cellulases lysates were added (100 pL) and incubated for one hour at RT. After washing with PBST, the wells were incubated for one hour at RT with a mouse antibody directed against the human c-Met intracellular Tyrosine-1234 residue (Cell Signaling), diluted 1: 1,000 in PBSC. The wells were washed again with PBST and incubated for one hour at RT with a goat anti-mouse Fc-HRP (Jackson) antibody diluted 1: 5,000 in PBSC. After washing with PBST, the reaction was visualized by a 30 minute incubation with 2,2'azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS: dilute one ABTS tablet in 50 mL of ABTS buffer [ Roche Diagnostics, Almere, Netherlands] on RT protected from light. Staining was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, Netherlands). Fluorescence at 405 nm was measured in a microtiter plate reader (Biotek Instruments, Winooski, USA). As shown in Figure 18, all 069 antibody mutants were able to induce inframodulation.
Example 26: Antibody dependent cell-mediated cytotoxicity (ADCC) [00297] MKN45 cells (purchased from RIKEN BioResource Center, Tsukuba, Japan, RCB1001) were harvested (5x10 ⁶ cells), washed (twice in PBS, 1,500 rpm, 5 min) and collected in 1 mL of RPMI 1640 medium supplemented with 10% bovine cosmic serum (CCS) (HyClone, Logan, UT, USA), to which 200 pCi ⁵¹ Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, Netherlands). The mixture was incubated in a shaking water bath for 1.5 hours at 37 ° C. After washing the cells (twice in PBS, 1,500 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted by trypan blue exclusion and diluted to a concentration of 1x10 ⁵
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119/121 cells / ml.
[00298] Meanwhile, peripheral blood mononuclear cells (PBMCs) were isolated from new leukocyte layers (Sanquin, Amsterdam, Netherlands) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspending cells in RPMI 1640 medium supplemented with 10% CCS, cells were counted by trypan blue exclusion and concentrated to 1x107 cells / mL.
[00299] For each ADCC experiment, 50 pL of ⁵¹ Cr-labeled MKN45 cells (5,000 cells) were preincubated with 15 pg / ml anti-cMet antibody in a total volume of 100 pL of RPMI medium supplemented with 10% of CCS in a 96-well microtiter plate. After 15 min in RT, 50 pL of PBMCs (500,000 cells) were added, resulting in an effective cell to target cell ratio of 100: 1. The maximum amount of cell lysis was determined by incubating ⁵¹ Cr-labeled MKN45 cells (5,000 cells) with 100 µl of 5% Triton-X100. The amount of spontaneous lysis was determined by incubation of 5,000 MKN45 cells labeled with ⁵¹ Cr in 150 pL of medium, without antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5,000 MKN45 cells with 500,000 PBMCs without antibody. Subsequently, the cells were incubated for 4 hours at 37 ° C, 5% CO2. The cells were centrifuged (1,200 rpm, 3 min) and 75 µl of supernatant was transferred to micronic tubes, after which the released ⁵¹ Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows:
[(sample cpm - Ab independent lysis cpm) / (maximum lysis cpm - spontaneous lysis cpm)] x 100%
Several publications have demonstrated the correlation between
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120/121 reduced core fucosylation and enhanced ADCC activity in vitro (Shields RL. 2002 JBC; 277: 26733-26740, Shinkawa T. 2003 JBC; 278 (5): 3466-3473). Figure 19 demonstrates that antibody 069 does not induce lysis of MKN45 cells by means of ADCC. However when nucleus fucosylation was reduced due to the presence of kifunensin during the production of mAb in HEK cells, antibody 069 was able to induce more than 30% lysis of MKN45 cells. In addition, lysis has already been observed at antibody concentrations below 0.01 pg / mL. Values shown are the average maximum release percentages of ⁵¹ Cr ± the standard deviation of a representative in vitro ADCC experiment with MKN45 cells. 069 low in fucose was produced in HEK 293 cells in the presence of kifunensin, resulting in ~ 99.5% non-nucleus fucosylation (ie no fucose). 069 high in fucose was produced in HEK 293 cells without kifunensin, resulting in ~ 2.11% non-nucleus fucosylation, as determined with high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) (data not shown ).
Example 27: Lack of binding of anti-c-Met antibodies in human peripheral blood cells [00300] For the purpose of resolving the binding of clone 069 in three types of cells (B cells, monocytes and granulocytes) present in blood peripheral, a binding assay was performed using FACS. Clone 069 fluorescently labeled was used to allow direct measurement in FACS without the use of secondary detection antibodies. The population of cells in the blood was identified in the assay using commercial fluorescent antibodies against specific markers on the cells of interest.
[00301] Peripheral blood from healthy volunteers (University Medical Center Utrecht) was diluted ten times in FACS buffer (PBS + 0.4% BSA + 0.02% NaN ₃ ) and incubated with conjugated anti-c-Met antibodies
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121/121 with Alexa ⁴⁸⁸ and FITC-conjugated anti-CD19, -CD16 and -CD14 antibodies (10 pg / mL final concentration) and phycoerythrin (PE)-labeled anti-CD19, -CD16 and -CD14 antibodies (BD Biosciences, San Jose CA) to identify cell populations (resp. B cells, granulocytes and monocytes) in a final volume of 100 pL. After 30 minutes at 4 ° C, the samples were centrifuged (300 g, 3 min), the supernatant was removed, erythrocytes were lysed by incubation (10 min, 4 ° C) with 200 pL of lysis-Ery solution (NH ₄ 155 mM Cl, 10 mM KHCO ₃ , 0.1 mM EDTA [H 7.4]), and samples were washed twice in FACS buffer. Samples were resuspended in 100 pL of FACS buffer and analyzed using a FACS Canto II (BD Biosciences).
[00302] Figure 20 is a representative FACS plot that demonstrates that 069 conjugated to Alexa ⁴⁸⁸ does not bind in a B cell population (CD19-PE ⁺ cells within the lymphocyte port). Binding of rituximab conjugated to Alexa ⁴⁸⁸ was used as a positive control. Binding in other cell populations was analyzed similarly and representative results for 1 of 3 donors are also plotted in Figure 21. Antibody 069-Alexa ⁴⁸⁸ did not bind to B cells, monocytes or granulocytes, while positive control antibodies demonstrated specific binding .

权利要求:
Claims (29)
[1]
1. Monoclonal antibody, characterized by the fact that it binds to a human c-Met, in which the antibody comprises:
a) a VH region comprising the sequences of CDR1, 2 and
3 of SEQ ID NO: 34, 35 and 36 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 38, 39 and 40, (024), or
b) a VH region comprising the sequences of CDR1, 2 and
3 of SEQ ID NO: 74, 75 and 76 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 78, 79 and 80, (062), or
c) a VH region comprising the sequences of CDR1, 2 and
3 of SEQ ID NO: 82, 83 and 84 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 86, 87 and 88, (064), or
d) a VH region comprising the sequences of CDR1, 2 and
3 of SEQ ID NO: 90, 91 and 92 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 94, 95 and 96, (068), or
e) a VH region comprising the sequences of CDR1, 2 and
3 of SEQ ID NO: 98, 99 and 100 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 102, 103 and 104, (069), or
f) a VH region comprising the CDR1 sequences,
[2]
2 and 3 of SEQ ID NO: 130, 131 and 132 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 134, 135 and 136, (181)
g) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 114, 115 and 116 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 118, 119 and 120 ( 098), or
h) a VH region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 122, 123 and 124 and a VL region comprising the sequences of CDR1, 2 and 3 of SEQ ID NO: 126, 127 and 128 ( 101).
2. Antibody according to claim 1, characterized by the fact that it comprises:
Petition 870190140759, of 12/27/2019, p. 9/18
2/7
a) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 37 (024)
b) a VH region comprising the sequence of SEQ ID NO: 73 and a VL region comprising the sequence of SEQ ID NO: 77 (062)
c) a VH region comprising the sequence of SEQ ID NO: 81 and a VL region comprising the sequence of SEQ ID NO: 85 (064)
d) a VH region comprising the sequence of SEQ ID NO: 89 and a VL region comprising the sequence of SEQ ID NO: 93 (068)
e) a VH region comprising the sequence of SEQ ID NO: 97 and a VL region comprising the sequence of SEQ ID NO: 101 (069)
f) a VH region comprising the sequence of SEQ ID NO:
113 and a VL region comprising the sequence of SEQ ID NO: 117 (098)
g) a VH region comprising the sequence of SEQ ID NO: 121 and a VL region comprising the sequence of SEQ ID NO: 125 (101)
h) a VH region comprising the sequence of SEQ ID NO: 129 and a VL region comprising the sequence of SEQ ID NO: 133 (181); or
i) a variant of any one of said antibodies, said variant preferably having at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.
[3]
Antibody according to either of claims 1 or 2, characterized in that the antibody binds in the SEMA domain of
Petition 870190140759, of 12/27/2019, p. 10/18
3/7 c-Met, preferably where the antibody is able to inhibit HGF binding in the SEMA domain with an IC 50 of less than 10 pg / ml, such as less than 2 pg / ml.
[4]
Antibody according to any one of the preceding claims, characterized in that the antibody binds to A431 cells with an EC50 of 10 nM or less, such as an EC50 of 2 nM or less.
[5]
5. Antibody according to claim 4, characterized in that the antibody is a bivalent antibody.
[6]
6. Antibody according to any one of the preceding claims, characterized in that the antibody binds to c-Met with an affinity constant (KD) of 20 nM or less, such as an affinity of 5 nM or less.
[7]
Antibody according to any one of the preceding claims, characterized in that the antibody inhibits the binding of HGF in the extracellular domain of c-Met, preferably wherein the antibody inhibits binding by more than 40%, such as more than 50%, p. more than 60%, e.g. more than 70%, e.g. more than 80%, e.g. more than 90%.
[8]
Antibody according to any one of the preceding claims, characterized in that the antibody is able to inhibit the viability of KP4 cells, preferably wherein the antibody is able to inhibit viability by more than 10%, such as more than 25%, p. eg more than 40%.
[9]
Antibody according to any one of the preceding claims, characterized in that the antibody is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1, K antibody.
[10]
10. Antibody according to any of the claims
Petition 870190140759, of 12/27/2019, p. 11/18
4/7 preceding, characterized by the fact that the antibody is conjugated to another group, such as a cytotoxic group, a radioisotope or a drug.
[11]
11. Antibody according to any one of the preceding claims, characterized by the fact that the antibody is an antibody deficient in effector function, e.g. a human stabilized IgG4 antibody, such as an antibody in which arginine at position 409 in the human IgG4 heavy chain constant region is replaced by lysine, threonine, methionine, or leucine, preferably lysine and / or in which articulation region comprises a Cys-Pro-Pro-Cys sequence.
[12]
12. Antibody according to any one of the preceding claims, characterized in that the antibody is a monovalent antibody.
[13]
13. Antibody according to claim 12, characterized by the fact that the monovalent antibody comprises:
(i) a variable region of an antibody according to claims 1 or 2 or a part that binds to the antigen of said region, and (ii) a CH region of an immunoglobulin or a fragment thereof comprising the CH2 and CH3 regions , in which the CH region or fragment thereof was modified in such a way that the region corresponding to the articulation region and, if the immunoglobulin is not an IgG4 subtype, other regions of the CH region, such as the CH3 region, do not comprise any residues of amino acids that are capable of forming disulfide bonds with an identical CH region or other stable covalent or non-covalent bonds inter-heavy chains with an identical CH region in the presence of a human polyclonal IgG.
[14]
14. Antibody according to any of the preceding claims, characterized by the fact that the antibody has been modified to make it less flexible, such as by mutations in the joint region.
[15]
Antibody according to claim 14, characterized
Petition 870190140759, of 12/27/2019, p. 12/18
5 due to the fact that the antibody is of the IgG1 subtype, and in which the joint region has been modified by:
(i) deletion of the articulation region of the EPKSCDKTHTCPPCP sequence (SEQ ID NO: 213) and its replacement by the IgG2 articulation region of the sequence: ERKCCVECPPCP (IgG1 articulationIgG2) (SEQ ID NO: 214);
(ii) deletion of position 220 so that the articulation region has the sequence of EPKSDKTHTCPPCP (IgG1 AC220) (SEQ ID NO: 215);
(iii) replacement of cysteine at position 220 by any other natural amino acid (X) so that the articulation region has the sequence of EPKSXDKTHTCPPCP (IgG1 C220X) (SEQ ID NO: 216);
(iv) deletion of the sequence joint region
EPKSCDKTHTCPPCP (UniBody IgG1) (SEQ ID NO: 213);
(v) deletion of the region of articulation of the sequence
EPKSCDKTHTCPPCP (SEQ ID NO: 213) and its replacement by the IgG3 articulation region of the sequence
ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDT PPPCPRCP (IgG1 joint-IgG3) (SEQ ID NO: 217); or (vi) replacement of threonine in position 223 by cysteine, and deletion of lysine in position 222 and threonine in position 225, so that the joint region has the sequence of EPKSCDCHCPPCP (IgG1 ΤΗ7Α6-9) (SEQ ID NO: 218 ).
[16]
16. Antibody according to claim 15, characterized by the fact that the joint region has been modified by replacing cysteine at position 220 by serine so that the modified joint region has the sequence of EPKSSDKTHTCPPCP (IgG1 C220S) (SEQ ID NO: 219).
[17]
17. Antibody according to any of the preceding claims, characterized by the fact that the antibody has been modified to
Petition 870190140759, of 12/27/2019, p. 13/18
6/7 reduce core fucosylation below 10%, as well as below 5%, as determined by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).
[18]
18. Antibody according to any of the preceding claims, characterized in that the antibody is a bispecific antibody, comprising a c-Met binding site, as defined in any of the preceding claims, and a second binding site. antigen having a different binding specificity.
[19]
19. Bispecific antibody according to claim 18, characterized in that the second antigen binding site has a binding specificity for a human effector cell, a human Fc receptor, a B cell receptor or an epitope of no overlap of c-Met.
[20]
20. Nucleotide sequence, characterized by the fact that it encodes the VH and VL regions of an antibody as defined in the claim
2.
[21]
21. Expression vector, characterized by the fact that it comprises a nucleotide sequence as defined in claim 20, the vector further encoding an operably linked constant region of a light chain, constant region of a heavy chain or both light and heavy chains of an antibody.
[22]
22. Recombinant prokaryotic host cell, characterized by the fact that it produces an antibody as defined in any of claims 1 to 19.
[23]
23. Pharmaceutical composition, characterized in that it comprises an antibody as defined in any one of claims 1 to 19 and a pharmaceutically acceptable carrier.
[24]
24. Antibody according to any one of claims 1 to 19, characterized by the fact that it is for use as a medicine.
[25]
25. Antibody according to any of the claims
Petition 870190140759, of 12/27/2019, p. 14/18
7/7
1 to 19, characterized by the fact that it is for use in the treatment of cancer, such as an HGF-dependent cancer or an HGF-independent cancer.
[26]
26. Method for producing an antibody as defined in any of claims 1 to 19, characterized in that said method comprises the steps of
a) culturing a host cell as defined in claim 22, and
b) purify the antibody from the culture medium.
[27]
27. Method for detecting the presence of c-Met in a sample, characterized by the fact that it comprises:
- contacting the sample with the antibody as defined in any of claims 1 to 19 under conditions that allow a complex to form between the antibody and c-Met; and
- analyze whether a complex has been formed.
[28]
28. Kit to detect the presence of c-Met in a sample, characterized by the fact that it comprises:
- an antibody as defined in any one of claims 1 to 19; and
- instructions for using the kit.
[29]
29. Use of the antibody as defined in any of claims 1 to 19, characterized in that it is for the manufacture of a cancer treatment drug.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-10-15| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-02-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-04-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 10/03/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US31262210P| true| 2010-03-10|2010-03-10|

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DKPA201000191|2010-03-10|

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DKPA201000862|2010-09-24|

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PCT/EP2011/053646|WO2011110642A2|2010-03-10|2011-03-10|Monoclonal antibodies against c-met|

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