combinations of c-met antibodies

专利摘要:
ANTIBODIES ANTI C-MET. The present invention relates to antibodies that specifically bind to the human c-Met receptor protein and that act as strict antagonists of hepatocyte growth factor (HGF) -mediated c-Met receptor and also inhibit the independent activation of Human c-Met protein HGF.
公开号:BR112013010688B1
申请号:R112013010688-3
申请日:2011-11-03
公开日:2020-11-17
发明作者:Anna Hultberg；Michael Saunders；Johannes DE HAARD；Els FESTJENS；Natalie DE JONGE；Paulo MICHIELI；Cristina Basilico；Torsten Dreier
申请人:Argenx Bvba；
IPC主号:

专利说明:

Technical Field
The present invention relates to product combinations comprising mixtures of antibodies and also multi-specific antibodies that bind to human c-Met and inhibit HGF independent activation of the c-Met receptor. Background
The tyrosine kinase receptor, c-Met, and its hepatocyte growth factor (HGF) ligand have become leading candidates for targeted cancer therapies. c-Met is the cell surface receptor for hepatocyte growth factor (HGF), also known as spreading factor. The c-Met receptor is a disulfide-linked heterodimer consisting of extracellular α and β chains. The a chain, heterodimerized to the amino terminal portion of the β chain, forms the main ligand binding site in the extracellular domain. The binding of HGF induces homodimerization and phosphorylation of the cMet receptor of two tyrosine residues (Y1234 and Y1235) within the catalytic site, regulating the activity of the kinase.
HGF-mediated activation of c-Met results in a complex genetic program referred to as "invasive growth", consisting of a series of physiological processes, including proliferation, invasion, and angiogenesis, which occurs under normal physiological conditions during embryonic and pathological development during oncogenesis. Signaling through c-Met promotes cell proliferation and survival through a variety of downstream effectors.
In tumor cells, activation of c-Met triggers diverse series of signaling cascades resulting in cell growth, proliferation, invasion and protection from apoptosis. The fundamental biological mechanisms for the origin of the c-Met tumor are typically achieved in three different ways: (a) with the establishment of the autocrine bonds of the HGF / c-Met; (b) via the overexpression of c-Met or HGF; and (c) in the presence of mutations activating the kinase in the sequence encoding the c-Met receptor. Expression of HGF and c-Met has been observed in tumor biopsies of most solid tumors, and c-Met signaling has been documented in a wide range of human malignancies, including bladder, breast, cervical, cervical cancers , gastric, head and neck, liver, lung, ovary, pancreatic, prostate, renal and thyroid.
Activation of c-Met by its ligand, HGF, can occur either in a paracrine or an autocrine manner. Paracrine activation can become pathological in the presence of abnormal HGF production. Autocrine activation occurs when tumor cells are expressed in an aberrant manner in both the HGF and its receptor. In addition, c-Met activation can occur independently of HGF, mediated by c-Met homodimerization.
A wide variety of human malignancies exhibit sustained C-Met stimulation, overexpression or mutation, including carcinomas of the breast, liver, lung, ovary, kidney and thyroid. Mutations activated in c-Met have been positively identified in patients with a particular hereditary form of papillary kidney cancer, directly implicating c-Met in human tumorigenesis. Aberrant signaling of the pathway signaling c-Met due to dysregulation of the c-Met receptor or over-expression of its ligand, HGF, has been associated with an aggressive phenotype. Extensive evidence that c-Met signaling is involved in the progression and spread of various cancers and an improved understanding of its role in the disease has generated considerable interest in c-Met and HGH as major targets in the development of cancer drugs (Eder et al, Clin Cancer Research; 15 (7); 2009).
A variety of antagonists of the c-Met pathway with potential clinical applications are currently under clinical investigation. Potential c-Met antagonists include monoclonal antibodies that block the interaction of c-Met with its HGF ligand. The most extensively described is the 5D5 anti-c-Met antibody generated by Genentech (WO96 / 38557). The 5D5 behaves as a potent agonist when added alone in several models and as an antagonist when used as a Fab fragment or antibody with one arm (MetMab).
WO 2009/007427 describes mouse monoclonal antibodies to c-Met and chimeric variants in which the antigen-binding domains of the mouse monoclonal antibody, or humanized variant thereof, are copulated to the human IgGl constant region. However, although the original mouse monoclonal antibody, 224G11, exhibits antagonistic activity without significant intrinsic agonist activity, the copulation of the 224G11 antigen-binding domains to human IgGl generated a chimeric form of 224G11 that exhibited some agonist activity associated with efficacy reduced antagonist. The agonist activity exhibited by the 224G11 chimeric form can be reversed by engineered point mutations in the human IgGl heavy chain hinge domain. In this engineered variant, several human amino residues in the hinge region are replaced by murine residues occurring in equivalent positions in the murine IgG1 sequence. The antagonistic activity of the C-Met receptor is restored in the resulting engineered variant, but the total structural and sequence homology for human antibodies is reduced as a result of the required mutations in the hinge region. In addition, at least one of the hypervariable loops in 224G11 adopts a canonical structure that is not found in the human antibody repertoire.
WO 2007/126799 describes complete human monoclonal antibodies to c-Met. These antibodies behave as antagonists of the interaction with HGF, but no data is presented regarding the intrinsic agonist activity of these antibodies or their ability to inhibit c-Met dimerization.
WO 2010/059654 also describes monoclonal c-Met antibodies. These antibodies are characterized by binding to the α-chain of human c-Met and induce the internalization of human c-Met on the cell surface. Description of the invention
It has now been observed that combinations (i.e., mixtures) of antibodies binding to human c-Met protein, and more specifically, combinations of two or more cMet antibodies that bind to distinct, non-overlapping epitopes in human nuclei. tpm advantageous properties mis are highly relevant for human therapeutic use. More particularly, it has been observed that such combinations of c-Met antibodies can produce potent inhibition of HGF independent activation of the human c-Met receptor. For certain combinations, the potency of inhibiting HGF-independent activation of the human c-Met receptor achieved with the combination is significantly more potent than that achieved using antibodies of the individual component of the combination in isolation. In addition, for certain combinations, the increase in potency to inhibit HGF independent activation of the human c-Met receptor is accompanied by a reduction in intrinsic agonist activity, as compared to antibodies to individual components present in the combination. It is for this reason that it has been proposed that combinations (mixtures) of two or more c-Met antibodies binding to distinct, non-overlapping epitopes, and also multi-specific antibodies in which the specificity of the component's antibodies are combined into a single molecule, are highly promising agents for triggering the c-Met receptor in human therapy.
The extracellular domain of c-Met is a highly complex structure, comprising several subdomains, including the low affinity binding site for HGF, the HIS 1 η narãn ei hinged evaHa nm ^ v ^ rr-i Sn hinge. Current perceptions in receptor biology suggest that c-Met, when bound to HGF to the low affinity binding site, undergoes a conformational change, allowing HGF binding to the high affinity binding site, followed by dimerization , activation and signaling of the receiver.
Without wishing to be bound by theory, it is assumed here that the combination of two or more antibodies, binding to non-overlapping epitopes on c-Met can be particularly successful if these antibodies prevent binding of HGF to both the binding site of low as well as high affinity of the HGF to the receptor. A product combination or composition comprising such antibodies can also be particularly effective if it also stereochemically prevents the conformational change caused by the binding of HGF to the low-affinity binding site of c-Met, required for the binding of HGF to the site of high affinity bond. This stereochemical impediment can be particularly effective if the binding of antibodies occurs in the two non-overlapping epitopes and effectively interferes with the dimerization of the receptor in an HGF-independent mode or by freezing the structure in a given conformation, or maintaining the
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For that reason, according to a first aspect of the invention, a product combination or composition comprising two or more antibodies or antigen-binding fragments thereof is provided, each of which binds to a human c-Met receptor protein in which at least at least two of said antibodies or antigen-binding fragments thereof bind to distinct, overlapping epitopes of the human c-Met protein, and in which the product combination or composition inhibits the HGF independent activation of the c-Met receptor protein human.
In one embodiment the combination or composition of the product can further inhibit HGF-dependent activation of the human c-Met receptor protein.
In another embodiment, the product combination or composition does not exhibit significant intrinsic agonist activity against the human c-Met receptor protein.
In another embodiment each of the two or more antibodies or antigen-binding fragments thereof in a combination or composition of the product is a strict antagonist of the HGF-mediated activation of the human c-Met receptor protein.
In another embodiment the combination or the HGF of the human c-Met receptor protein, and can behave as a strict antagonist of the HGF-mediated activation of the human c-Met receptor protein.
In one embodiment the combination or composition of the product comprises a first antibody or antigen-binding fragment that binds to an epitope within the PSI-IPT region of the human c-Met protein or to an epitope within the IPT region of protein c -Human met and a second antibody or antigen-binding fragment that binds to an epitope within the SEMA domain of the human c-Met protein.
In one embodiment of this combination or product composition the first antibody or antigen-binding fragment of the same blocks the binding of HGF to the high-affinity HGF binding site of the human c-Met protein and the second antibody or fragment of antigen binding blocks HGF binding to the low-affinity HGF binding site of human c-Met protein.
In a first embodiment of this combination or product composition the first antibody competes with the reference antibody 4 8A2 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 48A2, wherein the antibody of -P-i -1 / tom <n 4 rx z-. i nanziz} -. variable comprising the amino acid sequence shown as SEQ ID NO: 49 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 89. In this embodiment the second antibody is preferably an antibody that competes with the reference antibody 36C4 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 36C4, wherein the antibody reference 36C4 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 55.
In a second embodiment of this combination or product composition, the first antibody competes with the reference antibody 13E6 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the 13E6 reference antibody, where the reference antibody 13E6 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 46 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 57. In this embodiment the second antibody is preferably a cue antibody that competes with the reference antibody 20F1 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 20F1, where Reference 20F1 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 48 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 54.
In another embodiment the product composition or combination comprises a first antibody or antigen-binding fragment that binds to an epitope within the PSI-IPT region or the IPT region of the human c-Met protein and a second antibody or fragment of antigen binding that binds to an epitope within the PSI-IPT region or the IPT region of the human c-Met protein, in which the epitopes bound by the first and second antibodies, or antigen binding fragments thereof, are non-overlapping .
In a specific embodiment the first antibody competes with the reference antibody 4 8A2 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 4 8A2, where the antibody of reference 4 8A2 comprises a variable nested chain domain comprising the secretion of amino acids shown as SEQ ID NO: 49 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 89 and the second antibody competes with the antibody of reference 13E6 for binding to human c-Met protein or binds to the same epitope on human c-Met protein as the reference antibody 13E6, wherein reference antibody 13E6 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 46 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 57.
In another embodiment the product composition or combination comprises a first antibody or antigen-binding fragment that binds to an epitope within the SEMA domain of the human c-Met protein and a second antibody or antigen-binding fragment that binds to a distinct epitope within the SEMA domain of the human c-Met protein, in which the epitopes bound by the first and second antibodies, or antigen-binding fragments thereof, are non-overlapping.
In a first embodiment of this combination or product composition, the first antibody competes with the reference antibody 36C4 for binding to the human c-Met protein or links to the same epitope on the human c-Met protein as the reference antibody 36C4, in that reference antibody 36C4 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 55 and the second antibody competes with the reference 20F1 antibody for binding to human c-Met protein or binds to the same epitope on human c-Met protein as the 20F1 reference antibody, wherein the 20F1 reference antibody comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 48 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 54.
In a second embodiment of this combination or product composition the first antibody competes with the reference antibody 36C4 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 36C4, in that reference antibody 36C4 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain comprising the amino acid sequence shown as SEQ TΠwn. ç; t; csom mH snf * -5 Ho 34H7 reference for binding to human c-Met protein or binds to the same epitope on human c-Met protein as the 34H7 reference antibody, wherein the 34H7 reference antibody comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 77 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 78.
According to another aspect of the invention a multi-specific antibody is provided that binds to human c-Met protein, the multi-specific antibody comprising a first antigen binding region comprising a variable heavy chain domain in par with a domain of variable light chain and a second antigen binding region comprising a variable heavy chain domain paired with a variable light chain domain, wherein the first and second antigen binding region bind to different epitopes of the human c-Met protein , and in which the multi-specific antibody inhibits the HGF independent activation of the human c-Met receptor.
In one embodiment, the multispecific antibody additionally inhibits HGF-dependent activation of the human c-Met receptor.
Rm a Ho roa 1 i σaran n anti-i rrimn miilbi. does not exhibit significant intrinsic agonist activity against the human c-Met receptor.
In a specific embodiment at least one and preferably each of the antigen binding regions present in the multi-specific antibody is a strict antagonist of the HGF-mediated activation of the c-Met receptor, or is obtained from an antibody that is a strict antagonist of HGF-mediated activation of the c-Met receptor.
In an embodiment of the multispecific antibody the first antigen-binding region binds to an epitope within the PSI-IPT region of the human c-Met protein or to an epitope within the IPT region of the human cMet protein and the second binding region the antigen binds to an epitope within the SEMA domain of the human c-Met protein.
In one embodiment of this multispecific antibody the first antigen binding region can block the binding of HGF to the high affinity HGF binding site of the human c-Met protein and the second antigen binding region can block binding of the HGF for the low affinity HGF binding site of human c-Met protein.
In a first embodiment of this antibody, it is very likely to nipple the typical ion A antiσpnn is able to compete with the reference antibody 48A2 for binding to or binding to the human c-Met protein epitope on the human c-Met protein as the reference antibody 48A2, wherein the reference antibody 48A2 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 49 and a variable light chain domain comprising the sequence amino acid shown as SEQ ID NO: 89. In this embodiment the second antigen binding region is preferably an antigen binding region capable of competing with the reference antibody 36C4 for binding to human c-Met protein or that binds to the same epitope in human c-Met protein as the reference antibody 36C4, wherein the reference antibody 36C4 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 55 .
In one embodiment of this multispecific antibody the first antigen binding region competes with the reference antibody 13E6 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 13E6, in which variable heavy chain comprising the amino acid sequence shown as SEQ ID NO: 46 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 57. In this embodiment the second antigen-binding region is preferably an antigen-binding region that competes with the reference antibody 20F1 for binding to human c-Met protein or that binds to the same epitope in human c-Met protein as the reference antibody 20F1, wherein reference antibody 20F1 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 48 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 54.
In another embodiment of the multispecific antibody the first antigen-binding region binds to an epitope within the PSI-IPT region or the IPT region of the human c-Met protein and the second antigen-binding region binds to a distinct epitope within the PSI-IPT region or the IPT region of the human c-Met protein, in which the epitopes linked by the first and second antigen binding regions are non-overlapping.
In a particular embodiment of this antibody it competes with the reference antibody 48A2 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 48A2, wherein the reference antibody 48A2 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 49 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 89 and the second antigen binding region competes with the reference antibody 13E6 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 13E6, wherein the 13E6 reference antibody comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 46 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 57.
In another embodiment of the multispecific antibody the first antigen binding region binds to an epitope within the SEMA domain of the human c-Met protein and the second antigen binding region binds to a distinct epitope within the SEMA domain of the protein human cMet, in which the epitopes linked by the first and
In one embodiment of this multispecific antibody, the first antigen binding region competes with the reference antibody 36C4 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 36C4, in that reference antibody 36C4 comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 55 and the second binding region a antigen competes with the 20F1 reference antibody for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the 20F1 reference antibody, wherein the 20F1 reference antibody comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 48 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 54.
In another embodiment of this multispecific antibody the first antigen binding region competes with the reference antibody 36C4 for binding to the human c-Met protein or binds to the same epitope on the human c-Met protein as the reference antibody 36C4, in that variable heavy chain comprising the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 55 and the second antigen binding region competes with the reference antibody 34H7 for binding to human c-Met protein or binds to the same epitope in human c-Met protein as the 34H7 reference antibody, wherein the 34H7 reference antibody comprises a variable heavy chain domain comprising the amino acid sequence shown as SEQ ID NO: 77 and a variable light chain domain comprising the amino acid sequence shown as SEQ ID NO: 78.
The individual antibodies included in a combination or composition of the product provided here, or the antigen binding regions present in the multi-specific antibody provided here, can each specifically bind to a human c-Met protein and exhibit at least two or all of the following three properties: (a) be a strict antagonist of the HGF-mediated activation of the human c-Met protein, (b) inhibit the HGF-independent activation of the human c-Met protein, and (c) do not induce negative regulation cell.
In one embodiment, the individual antibodies included in a combination or composition of the product provided here, or the antigen binding regions present in the multi-specific antibody provided here, can specifically bind to a human c-Met protein and exhibit the properties (a) be an antagonist of HGF-mediated activation of human c-Met protein, (b) inhibit HGF-independent activation of human c-Met protein.
In one embodiment, the individual antibodies included in a combination or composition of the product provided here, or the antigen binding regions present in the multi-specific antibody provided here, can specifically bind to a human c-Met protein and exhibit the properties (a) be a strict antagonist of the HGF-mediated activation of the human c-Met protein, and (c) not induce significant negative regulation of the human c-Met protein on the cell surface.
In one embodiment, the individual antibodies provided here, or the antigen-binding regions present in the multi-specific antibody provided here, can specifically bind to a human c-Met protein and exhibit the following properties: (b) inhibit activation independent of the human c-Met protein HGF, and (c) does not induce significant negative regulation of the human c-Met protein on the cell surface.
In one embodiment, the individual antibodies included in a combination or composition of the product provided here, or the antigen binding regions present in the multi-specific antibody provided here, can specifically bind to a human c-Met protein and display all of the following properties: (a) be a strict antagonist of the HGF-mediated activation of the human c-Met protein, (b) inhibit the HGF-independent activation of the human c-Met protein, and (c) not induce significant negative regulation of human c-Met protein on the cell surface.
The individual antibodies included in a multi-specific combination provided herein, can comprise a hinge region having the complete human sequence. The individual antibodies in a product combination or composition, or the multi-specific antibody, also have high human homology, as defined here.
The individual antibodies included in a combination or composition of the product provided herein, or the multi-specific antibody provided here can be any of, a monoclonal antibody, a fully human monoclonal antibody, or a humanized monoclonal antibody. The individual antibodies included in a combination or composition of the product provided herein may exhibit bivalent binding to the human c-Met protein.
The multi-specific antibody provided herein can be a bi-specific antibody.
In particular embodiments, the individual antibodies included in a combination or composition of the product, or the antigen-binding regions present in the multi-specific antibody, can comprise a variable heavy chain (VH) domain and a variable light chain domain (VL), in which the VH and VL domains, or one or more CDRs thereof, are derived from camelids.
In a particular embodiment, the antibodies ■ λ to η QTM1 im-a -í ri as ai i -í z-, ai z- z ^ z ^ product, or the antigen-binding regions present in the multi-antibody specific, they can comprise VH and VL llama domains, or germline strain variants of the VH and VL domains. This antibody, or antigen-binding fragment, may also exhibit "high human homology", as defined here. The individual antibodies included in a combination or composition of the product, or the multispecific antibody, can each be chimeric antibodies containing VH and VL domains that are derived from camelids, or humanized or germline-derived variants thereof, fused to domains that are constant to human antibodies , in particular human IgG1, IgG2, IgG3 or IgG4. These chimeric antibodies can include a hinge region having the complete human sequence, as defined here.
In the following section, preferred antibodies, or antigen-binding regions thereof, for inclusion in product combinations or compositions, or the multi-specific antibodies provided here will be further defined with respect to structural characteristics: (A) 48A2, 4 8A2 variants and antibodies / antigen-binding regions that bind to the same epitope in human c-Met as the reference antibody 48A2 product, or the multi-specific antibody, which comprises at least one antibody or antigen-binding region that binds to an epitope within the PSI-IPT region of the human c-Met, this antibody or antigen-binding region may be 48A2, or a germinalized strain or affinity variant thereof, or may be an antibody or a binding region antigen that competes with the reference antibody 4 8A2 for binding to human c-Met or that binds to the same epitope on human c-Met as the reference antibody 48A2.
The c-Met antigen binding site in the reference antibody 48A2 is provided in pairs of a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 49 and a variable light chain domain having the amino acid sequence shown such as SEQ ID NO: 89.
Reference antibody 48A2 showed that it binds to an epitope within the 523 peptide sequence Consequently, it is preferred to use 48A2 variants or competing antibodies / peptide-binding regions, reaching the PSI-IPT1 regions of human c-Met. Variants of 48A2 or competing antibodies / antigen-binding regions can block the binding of HGF to the high-affinity HGF binding site in human c-Met protein.
Preferred embodiments of 48A2 and variants of 4 8A2 for use in the product combination or composition, or as components of the multi-specific antibody are defined below by reference to structural aspects:
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein the variable heavy chain CDR3 sequence is SEQ ID NO: 15 or sequence variant thereof where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to the human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 15 or variant of the sequence thereof, -
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NO: XX3 [RIDPEXxGGTKYAQKFQG] where, X! is any amino acid, preferably D, N or E; and or variant of the sequence thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: XX6 [X1X2X3ID], or sequence variant thereof, wherein, Xi is any amino acid, preferably M or N, X2 is any amino acid, preferably N or Y, X3 is any amino acid, preferably S or V; and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 15 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 14 or sequence variant thereof or SEQ ID NO: 85 or variant sequence thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 13 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: YY1 [QQGXISFPX2X3], or sequence variant thereof, where Xi is any amino acid, preferably Y or W; X2 θ any amino acid, preferably Y or L; X3 is any amino acid, preferably T or S; the variable light chain CDR2 sequence is SEQ ID NO: YY3 [WASXjRES], or sequence variant thereof, where Xi is any amino acid, preferably I or T; and the variable light chain CDR1 sequence is SEQ ID NO: YY5 [KSSQSVLXIX2 X3N X4K X5YLA], OR sequence variant thereof, where Xi is any amino acid, preferably W, L or F; X2 is any amino acid, preferably R or S; X3 is any amino acid, preferably S or P; X4 is any amino acid, preferably Q or H; X5 is any amino acid, preferably N or S
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is selected from among the group consisting of SEQ ID NO: 87 or its sequence variant, SEQ ID NO: 24 or its sequence variant, SEQ ID NO: 139 or its sequence variant, and SEQ ID NO: 141 or sequence variant therein, wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is selected from among the group consisting of SEQ ID NO: 87 or sequence variant thereof, SEQ ID NO: 139 or variant sequence thereof, and SEQ ID NO: 141 or variant sequence thereof; the variable light chain CDR2 sequence is SEQ ID NO: 23 or variant of the sequence thereof or SEQ ID NO: 26 or among the group consisting of SEQ ID NO: 86 or variant of the sequence thereof, SEQ ID NO: 137 or sequence variant thereof, SEQ ID NO: 138 or sequence variant thereof, SEQ ID NO: 140 or sequence variant thereof, SEQ ID NO: 142 or sequence variant thereof, and SEQ ID NO: 143 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, where the variable light chain CDR3 sequence is SEQ ID NO: 24 or variant of the sequence thereof; the variable light chain CDR2 sequence is SEQ ID NO: 23 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 22 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to the human c-Met receptor protein, the antibody or antigen binding region comprising a variable domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 15 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 14 or sequence variant thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 13 or sequence variant thereof, the variable light chain CDR3 sequence is SEQ ID NO: 87 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 23 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 86 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, where: the CDR3 sequence variable heavy chain is SEQ ID NO: 15 or sequence variant thereof; the variable heavy chain CDR1 sequence is SEQ ID NO: 13 or sequence variant thereof; and the variable light chain domain includes a combination of the selected CDRs from the following: (i) the variable light chain CDR3 sequence is SEQ ID NO: 24 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 23 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 22 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (ii) the variable light chain CDR3 sequence is SEQ ID NO: 87 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 26 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 137 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (iii) the variable light chain CDR3 sequence is SEQ ID NO: 139 or sequence variant thereof; the variable light chain CDR1 sequence is SEQ ID NO: 138 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (iv) the variable light chain CDR3 sequence is SEQ ID NO: 141 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 26 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 140 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (v) the variable light chain CDR3 sequence is SEQ ID NO: 141 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 26 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 142 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (vi) the variable light chain CDR3 sequence is SEQ ID NO: 87 or sequence variant thereof; the variable light chain CDR1 sequence is SEQ ID NO: 86 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (vii) the variable light chain CDR3 sequence is SEQ ID NO: 87 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 26 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 143 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable heavy chain domain comprising a VH sequence with at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity, for an amino acid sequence selected from the group consisting of: SEQ ID NO: 49, 108, 110, 112, 114, 116, 118
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable heavy chain domain comprising a VH amino acid sequence selected from the group consisting of: SEQ ID NO: 49, 108, 110, 112, 114, 116, 118 and 120.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable light chain domain comprising a V Kappa sequence with at least 75% of the sequence identity, or at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, for an amino acid sequence selected from the group consisting of SEQ ID NO: 52, 89, 109, 111, 113, 115, 117, 119, 121, 149, 150, 151, 152, 153, 154, 155, 156 and 157.
An antibody or antigen binding region that is or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable light chain domain comprising a V Kappa amino acid sequence selected from the group consisting of SEQ ID NO: 52, 89, 109, 111, 113, 115, 117, 119, 121, 149, 150, 151, 152, 153, 154, 155, 156 and 157.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable heavy chain domain comprising a VH sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity for an amino acid sequence selected from the group consisting of: SEQ ID NO: 49, 108, 110, 112, 114, 116, 118 and 120 and the variable light chain domain comprising a V Kappa sequence with at least 75% of the identity of the sequence, or at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity for a selected amino acid sequence from group consisting of S EQ ID NO: 52, 89, 109, 111, 113, 115, 117, 119, 121, 149, 150, 151, 152, 153, 154, 155, 156 and 157.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 49, or a humanized or affinity variant thereof, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 52 or the amino acid sequence shown as SEQ ID NO: 89 or a humanized variant or its affinity.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain comprising a VH sequence with at least 85% sequence identity , or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity to SEQ ID NO: 49, and a variable light chain domain (VL) comprising a sequence V TZ m * 7 C S'o ibn rl oo om icnriη o 1 at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90% of the sequence identity , or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity to the amino acid sequence shown as SEQ ID NO: 52 or the amino acid sequence shown as SEQ ID NO: 89 .
This antibody, or antigen-binding region can comprise heavy chain CDRs that are identical to CDR1, CDR2 and CDR3 of SEQ ID NO: 49 and light chain CDRs that are identical to CDR1, CDR2 and CDR3 of SEQ ID NO: 89 or CDR1, CDR2 and CDR3 of SEQ ID NO: 52, although exhibiting variation in the amino acid sequence within the framework regions.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 49, or a humanized or affinity variant thereof, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 89 or a humanized or affinity variant thereof.
An antibody or antigen-binding region that would 1 -rro oo η in the r-Mob ^ iirriar ar b T r or antigen-binding region being a germline variant or affinity variant of the reference antibody 48A2, referred to variant comprising: (a) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 108, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 109 ; OR (b) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 110, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 111; OR (c) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 112, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 113; or (d) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 114, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ (e) a domain variable heavy chain (VH) comprising the amino acid sequence shown as SEQ ID NO: 116, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 117; OR (f) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 118, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 119; or (g) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 120, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 121; OR (h) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 49, and a variable light chain (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: : 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156 and SEQ ID NO: 157 .
These 48A2 variant antibodies, or combination binding regions, of a VH domain, defined by reference to a specific amino acid sequence, and a VL domain (V Kappa), also defined by reference to a specific amino acid sequence. For each specific VH / VL combination listed, this definition should be taken to include antibodies, or antigen-binding regions, formed by combining a VH domain having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the sequence identity for the specified VH amino acid sequence and a VL domino having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 97%, or at least 99% of the sequence identity for the specified VL amino acid sequence. In each case the VH and VL domains defined by the% sequence identity for the VH and VL amino acid sequence can retain CDR sequences identical to those present in the specified VH and VL amino acid sequence, while exhibiting variation in the amino acid sequence within the regions of the framework. (B) 36C4, 36C4 variants and antibodies / antigen binding regions that bind to the same epitope in human c-Met as the reference antibody 36C4
In embodiments of the oTodiibo combination or composition. or sntirnrnn mnl hi-Asnp.cí f i oo. cniA rnmnrppTiHp at least one antibody or antigen-binding region that binds to an epitope within the SEMA domain of human c-Met, this antibody or antigen-binding region can be 36C4, or a germline strain or affinity variant of the same, or it may be an antibody or antigen binding region that competes with the reference antibody 36C4 for binding to the human c-Met protein or binds to the same epitope on the human c-Met as the reference antibody 36C4.
The c-Met antigen binding site on the 36C4 reference antibody is provided in pairs of a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 51 and a variable light chain domain having the amino acid sequence shown such as SEQ ID NO: 55.
The reference antibody 36C4 showed that binds to an epitope within the SEMA domain of human c-Met, and more specifically an epitope within 98VDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCWSAL GAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETK-199 peptide (SEQ ID NO: 181) of human c-Met. The 36C4 or 36C4 variant antibody or antigen binding region can also bind to an epitope within this peptide region of the SEMA domain of
The region of the SEMA domain contained with the 98VDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCWSAL GAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETK-199 peptide is a significant sign of the HG-link is already meaningful because it is a HG link. The variant 36C4 or 36C4 antibody or antigen binding region can block the binding of HGF to the HGF binding site of the human c-Met protein by virtue of binding to an epitope within this region of the SEMA domain.
Preferred embodiments of 3604 and 36C4 variants for use in the product combination or composition, or as components of the multi-specific antibody are as defined below by reference to structural aspects:
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain where the variable heavy chain CDR3 sequence is SEQ ID NO : 21 or sequence variant thereof where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody <-> n lcrã to linarãn to amphyrres nnmnreenHendn nm dnminin variable heavy chain where the variable heavy chain CDR3 is SEQ ID NO: 21 or sequence variant thereof; the variable heavy chain CDR2 sequence is SEQ ID NO.-XX2 [VIAYDGSTXiYSPSLKS] or sequence variant thereof, where Xi is any amino acid, preferably Y or D; and the variable heavy chain CDR1 sequence is SEQ ID NO: XX5 [X1NYYX2WS], or sequence variant thereof, where Xi is any amino acid, preferably G or T, X2 is any amino acid, preferably A or Y; and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 21 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 20 or sequence variant thereof or SEQ ID NO: 83 or NO: 19 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: YY2 [ASYRXIX2X3X4X5X6V], or sequence variant thereof, where Xi is any amino acid, preferably S, I, R or T; X2 is any amino acid, preferably A, S, T or R; X3 is any amino acid, preferably N or T; X4 is any amino acid, preferably N, D, R or K; X5 is any amino acid, preferably A, V, Y, N or H; X6 is any amino acid, preferably V, A, S or G; the variable light chain CDR2 sequence is SEQ ID Xi is any amino acid, preferably D, A or E, X2 is any amino acid, preferably N or S, X3 is any amino acid, preferably R, Y or K, X4 is any amino acid, preferably A, or P; and the. variable light chain CDR1 sequence is SEQ ID NO: YY6 [X1GX2X3X4X5X6GX7X8X9YX10S], or sequence variant thereof, where Xi is any amino acid, preferably A or T; X2 is any amino acid, preferably T or S; X3 is any amino acid, preferably S or N; X4 is any amino acid, preferably S or T; X5 is any amino acid, preferably D or N; X6 is any amino acid, preferably V or I; X is any amino acid, preferably Y, G, D or N; Xs is any amino acid, preferably G or Y; X9 is any amino acid, preferably N or Y; X10 θ any amino acid, preferably V or L, and where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody to the variable light chain CDR3 sequence is selected from the group consisting of SEQ ID NO: 33 or sequence variant, SEQ ID NO: 145 or its sequence variant, SEQ ID NO: 146 or its sequence variant, SEQ ID NO: 147 or its sequence variant, and SEQ ID NO: 148 or its sequence variant, where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is selected from among the group consisting of SEQ ID NO: 33 or variant of the sequence thereof, SEQ ID NO: 145 or variant of the sequence thereof, SEQ ID NO: 146 or variant of the sequence thereof, SEQ ID NO: 147 or variant of the sequence same, and SEQ ID NO: 148 or variant of the sequence thereof; the variable light chain CDR2 sequence is SEQ ID NO: 32 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 31 or sequence variant thereof, or SEQ ID NO: 144 three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, where: the CDR3 sequence variable heavy chain is SEQ ID NO: 21 or sequence variant thereof; the variable heavy chain CDR2 sequence is selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 83 and SEQ ID NO: 84 or sequence variant thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 19 or sequence variant thereof; and the variable light chain domain includes a combination of the selected CDRs from the following: (i) the variable light chain CDR3 sequence is SEQ ID NO: 33 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 32 or variant of the sequence thereof; the variable light chain CDR1 sequence is SEQ ID NO: 31 or sequence variant thereof, (ii) the variable light chain CDR3 sequence is SEQ ID NO: 145 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 32 or variant of the sequence thereof; the variable light chain CDR1 sequence is SEQ ID NO: 144 or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (iii) the variable light chain CDR3 sequence is SEQ ID NO: 146 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 32 or variant of the sequence thereof; the variable light chain CDR1 sequence is SEQ ID NO: 31 or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; or (iv) the variable light chain CDR3 sequence is SEQ ID NO: 147 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 32 or variant of the sequence thereof; the variable light chain CDR1 sequence is SEQ ID NO: 144 or sequence variant thereof, three amino acid substitutions in the cited sequence; or (v) the variable light chain CDR3 sequence is SEQ ID NO: 148 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 32 or variant of the sequence thereof; the variable light chain CDR1 sequence is SEQ ID NO: 144 or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises a VH sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% identity from sequence to a sequence selected from the group consisting of: SEQ ID NO: 51, 88, 92, 94, 96 and 98.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the variable antibody, wherein the variable heavy chain domain comprises a VH amino acid sequence selected from the group consisting of: SEQ ID NO: 51, 88, 92, 94, 96 and 98.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain light variable comprises a V Lambda sequence with at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity for an amino acid sequence selected from the group consisting of SEQ ID NO: 55, 93, 95, 97, 99, 158, 159, 160, 161, 162, 163 and 164 .
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain light variable comprises a sequence of amino acids V Lambda col-rlonbro <-> rrrnnn rnnαi sti nHn rich çpf) TT> MD • R 9 9 7 95, 97, 99, 158, 159, 160, 161, 162, 163 and 164 .
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises a VH sequence with at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity sequence for a sequence selected from the group consisting of: SEQ ID NO-.51, 88, 92, 94, 96 and 98, and the variable light chain domain comprises a V Lambda sequence with at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity for an amino acid sequence selected from the group consisting of SEQ ID NO: 55, 93 , 95, 97, 99, 158, 159, 160, 161, 162, 163 and 164.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable oarloi = relapse domain ai> m Hnmírrm raHeifl 1 variable, where the domain of variable heavy chain comprises the amino acid sequence shown as SEQ ID NO: 51 or SEQ ID NO: 88 or a humanized or affinity variant thereof, and the variable light chain domain comprises the amino acid sequence shown as SEQ ID NO: 55 , or a humanized or affinity variant thereof.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises a VH sequence with at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity sequence for the amino acid sequence shown as SEQ ID NO: 51 or SEQ ID NO: 88, and the variable light chain domain comprises a V Lambda sequence with at least 80% of the sequence identity, or at least 85% of the sequence identity sequence, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity for the sequence This antibody, or antigen binding region can understand heavy chain CDRs that are id emphasis on CDR1, CDR2 and CDR3 of SEQ ID NO: 51 or CDR1, CDR2 and CDR3 of SEQ ID NO: 88 and light chain CDRs that are identical to CDR1, CDR2 and CDR3 of SEQ ID NO: 55, although showing variation amino acid sequence within the framework regions.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, wherein the antibody or antigen-binding region is a variant of germline lineage or affinity of the 36C4 antibody, said variant comprising: (a ) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 92, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 93; or (b) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 94, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 95; OR (c) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 97; OR (d) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 98, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 99; OR (e) a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 88, and a variable light chain (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: : 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 164 .
These variant 36C4 antibodies, or antigen-binding regions, are identified as comprising a combination of a VH domain, defined by reference to a specific amino acid sequence, and a VL domain (V Kappa), defined also by reference to a sequence of specific amino acids. For each specific VH / VL combination listed, should this definition be taken to include antibodies, or antigen binding regions, formed by combining a VH domain having at least npln mpnns 9C)% nsln mpnn « 9 ^%. npln mpnnç; 97% r> n at least 99% of the sequence identity for the specified VH amino acid sequence and a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% of the sequence identity for the specified VL amino acid sequence. In each case, the VH and VL domains defined by the% sequence identity for the VH and VL amino acid sequences can retain CDR sequences identical to those present in the specified VH and VL amino acid sequences, while exhibiting variation in the amino acid sequence within the regions of the framework. (C) Antibodies / antigen-binding regions that bind to the same epitope in human c-Met as the reference antibody 20F1
In embodiments of the combination or composition of the product, or the multi-specific antibody, which comprises at least one antibody or antigen-binding region that binds to an epitope within the SEMA of the human c-Met, this antibody or region of antigen binding may be 20F1, or a variant of germline or affinity variant thereof, or it may be an antibody or antigen binding region that competes with the reference antibody 20F1 for binding to human c-Met or alloy reference 20F1.
The c-Met antigen-binding site in the reference antibody 20F1 is provided in pairs of a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 48 and a variable light chain domain having the amino acid sequence shown as SEQ ID NO: 54s.
Preferred embodiments of variants 20F1 and 20F1 for use in the combination or composition of the product, or as components of the multi-specific antibody are as defined below by reference to structural aspects:
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain where the variable heavy chain CDR3 sequence is SEQ ID NO : 12 or sequence variant thereof where the sequence variant comprises one, two or three amino acid substitutions in the aforementioned sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain where z-r iA z- »4 / I • = »A -» C * T7r> TFl NO: 12 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO-.XX2 [VIAYDGSTXxYSPSLKS] or sequence variant thereof, where Xi is any amino acid, preferably Y or D; and the variable heavy chain CDR1 sequence is SEQ ID NO: XX5 [X] NYYX2WS], or sequence variant thereof, where Xi is any amino acid, preferably G or T, and X2 is any amino acid, preferably A or Y ; wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 12 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 11 or sequence variant thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 10 or sequence variant thereof, and wherein the sequence variant comprises one, two or
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: YY2 [ASYRXxX2X3X4X5X6V], or sequence variant thereof, where Xi is any amino acid, preferably S, I, R or T; X2 θ any amino acid, preferably A, S, T or R; X3 is any amino acid, preferably N or T; X4 is any amino acid, preferably N, D, R or K; X5 is any amino acid, preferably A, V, Y, N or H; X6 is any amino acid, preferably V, A, S or G; the variable light chain CDR2 sequence is SEQ ID NO: YY4 [Xiyx2X3RX4S], or sequence variant thereof, where Xx is any amino acid, preferably D, A or E, X2 is any amino acid, preferably N or S, lower am i nná ci Hn nrpfpri vpl mpntp PY nil K. X4 is any amino acid, preferably A, or P; and the variable light chain CDR1 sequence is SEQ ID NO: YY6 [XiÇp ^ XjX ^ sXgGXvXgXgYXioS], or sequence variant thereof, where Xi is any amino acid, preferably A or T; X2 is any amino acid, preferably T or S; X3 is any amino acid, preferably S or N; X4 is any amino acid, preferably S or T; X5 is any amino acid, preferably D or N; X6 is any amino acid, preferably V or I; X7 is any amino acid, preferably Y, G, D or N; X8 is any amino acid, preferably G or Y; X9 is any amino acid, preferably N or Y; Xio is any amino acid, preferably V or L in which the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: 30 or variant of the sequence of the same in which the variant of the semencium romnrpp.nde »one. two nu trp.s subst i t.ui rõp.s ida amino acids in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: 30 or variant of the sequence thereof; the variable light chain CDR2 sequence is SEQ ID NO: 29 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 28 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, where: the CDR3 sequence variable heavy chain is SEQ ID NO: 12 or sequence variant thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 11 or sequence variant thereof; and the variable light chain CDR3 sequence is SEQ ID NO: 30 or variant of the sequence thereof; the variable light chain CDR2 sequence is SEQ ID NO: 29 or variant of the sequence thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 28 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises a VH sequence with at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity sequence, for the amino acid sequence shown as SEQ ID NO: 48.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable rnmnropndo aomiênria ami nná i Hnc VW mnqbrarla r'nmr'i QP.O ID NO: 48.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain light variable comprises a V Lambda sequence with at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity for the amino acid sequence shown as SEQ ID NO: 54.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain light variable comprises the V Lambda amino acid sequence shown as SEQ ID NO: 54.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a domain comprises the amino acid sequence shown as SEQ ID NO: 48 or a humanized variant or of affinity thereof, and the variable light chain domain comprises the amino acid sequence shown as SEQ ID NO: 54, or a humanized or affinity variant thereof.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises a VH sequence with at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity sequence for the amino acid sequence shown as SEQ ID NO: 48, and the variable light chain domain comprises a V Lambda sequence with at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least 90 % sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, for the amino acid sequence shown as SEQ ID NO: 54. (D) Antigen-binding antibodies / regions that bind to the same eoeton in human c-Met as the 13E6 reference horn
In embodiments of the combination or composition of the product, or the multi-specific antibody, which comprises at least one antibody or antigen binding region that binds to the same epitope within the PSI-IPT region of the human c-Met, this antibody or antigen-binding region can be 13E6, or a germline variant or affinity variant thereof, or it can be an antibody or antigen-binding region that competes with the reference antibody 13E6 for binding to human c-Met or that binds to the same epitope on human c-Met as the reference antibody 13E6
The c-Met antigen binding site in the reference antibody 13E6 is provided by pairs of a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 46 and a variable light chain domain having the amino acid sequence shown such as SEQ ID NO: 57.
Preferred embodiments of variants 13E6 and 13E6 for use in the product combination or composition, or as components of the multi-specific antibody are as defined below by reference to structural aspects:
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein the variable heavy chain CDR3 sequence is SEQ ID NO: 6 or sequence variant thereof where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 6 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: XXI [XIX2X3X4X5X6X7X8TYYAESMK] or sequence variant thereof, where Xi is any amino acid, preferably T or A; X2θ any amino acid, preferably I, X3 is any amino acid, preferably S or N; X4 is any amino acid, preferably W, X5 is any amino acid, preferably N, X6 is any amino acid, preferably D or G; X7 is any amino acid, preferably I, G or S; and X8 is any amino acid, preferably N or S; that Xi is any amino acid, preferably D or S, X2 is any amino acid, preferably A or V, and X3 is any amino acid, preferably T, N or S; wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 6 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 5 or sequence variant thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 4 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: YY2 rASYRXnXQX ^ XdXqXfiV], or sequence variant thereof / in <Iue Xi is any amino acid, preferably S, I, R or T; Xz θ any amino acid, preferably A, S, T or R; X3 is any amino acid, preferably N or T; X4 is any amino acid, preferably N, D, R or K; X5 is any amino acid, preferably A, V, Y, N or H; Xe is any amino acid, preferably V, A, S or G; the variable light chain CDR2 sequence is SEQ ID NO: YY4 [XIVX2X3RX4S], or sequence variant thereof, where Xi is any amino acid, preferably D, A or E, X2 is any amino acid, preferably N or S, X3 is any amino acid, preferably R, Y or K, X4 is any amino acid, preferably A, or P; the variable light chain CDR1 sequence is SEQ ID NO.-YY6 [X1GX2X3X4X5X6GX7X8X9YXioS], or sequence variant thereof, where Xi is any amino acid, preferably A or T; X3 is any amino acid, preferably S or N; X4 is any amino acid, preferably S or T; X5 is any amino acid, preferably D or N; X6 is any amino acid, preferably V or I; X7 is any amino acid, preferably Y, G, D or N; X8 is any amino acid, preferably G or Y; Xg is any amino acid, preferably N or Y; Xio is any amino acid, preferably V or L, where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, where the variable light chain CDR3 sequence is SEQ ID NO: 39 or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID the variable light chain CDR2 sequence is SEQ ID NO: 38 or sequence variant thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 37 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, where: the CDR3 sequence variable heavy chain is SEQ ID NO: 6 or sequence variant thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 5 or sequence variant thereof; where the variable heavy chain CDR1 sequence is SEQ ID NO: 4 or sequence variant thereof, the variable light chain CDR3 sequence is SEQ ID NO: 39 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 38 or sequence variant thereof; and three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable heavy chain domain comprising a VH sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity for the amino acid sequence shown as SEQ ID NO: 46.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable heavy chain domain comprising the VH amino acid sequence shown as SEQ ID NO: 46.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable light chain domain comprising a V Lambda sequence with at least 80% of the identity of the côn lônn -ia on noln mαnnα RRSr from η donf-i da do da comienr'i to nn at least 90% of the sequence identity, or at least 95% of the identity of the sequence, or at least 97%, 98% or 99% of the sequence identity for the amino acid sequence shown as SEQ ID NO: 57.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, the variable light chain domain comprising the V Lambda amino acid sequence shown as SEQ ID NO: 57.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain (VH) domain comprising the amino acid sequence shown as SEQ ID NO: 46, or a humanized or affinity variant thereof, and a variable light chain (VL) domain comprising the amino acid sequence shown as SEQ ID NO: 57 or a humanized or affinity variant thereof.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a domain with at least 85% of the sequence identity, or at least 90% of the identity of the sequence, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity for the amino acid sequence shown as SEQ ID NO: 46, and a variable light chain domain (VL ) comprising a V Lambda sequence with at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97 %, 98% or 99% of the sequence identity, for the amino acid sequence shown as SEQ ID NO: 57. (E) Antigen-binding antibodies / regions that bind to the same epitope in human c-Met as the reference antibody 34H7
In embodiments of the combination or composition of the product, or the multi-specific antibody, which comprises at least one antibody or antigen-binding region that binds to an epitope within the SEMA of the human c-Met, this antibody or region of antigen binding may be 34H7, or a variant of germline or affinity variant thereof, or it may be an antibody or antigen binding region that competes with the reference antibody 34H7 for binding to human c-Met or alloy reference 34H7.
The c-Met antigen binding site on the 34H7 reference antibody is provided by pairs of a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 77 and a variable light chain domain having the amino acid sequence shown such as SEQ ID NO: 78.
Preferred embodiments of variants 34H7 and 34H7 for use in the combination or composition of the product, or as components of the multi-specific antibody are defined below by reference to structural aspects:
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain where the variable heavy chain CDR3 sequence is SEQ ID NO : 73 or sequence variant thereof where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain, in which: ■ the ooimiânr'-i cs r'HD 'J r * «cs -í cs vs / cs oo z4 cs Trcvi OTrol o QTTT'» TH NO: 73 or its sequence variant; the variable heavy chain CDR2 sequence is SEQ ID NO: 72 or sequence variant thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 71 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, where the variable light chain CDR3 sequence is SEQ ID NO: 76 or sequence variant thereof where the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: 76 or variant of the sequence thereof; the variable light chain CDR2 sequence is SEQ ID NO: 75 or sequence variant thereof; and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence.
An antibody or antigen binding region that binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain, wherein: the variable heavy chain CDR3 sequence is SEQ ID NO: 73 or variant of the sequence thereof; the variable heavy chain CDR2 sequence is SEQ ID NO: 72 or sequence variant thereof; and the variable heavy chain CDR1 sequence is SEQ ID NO: 71 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence; and a variable light chain domain, wherein: the variable light chain CDR3 sequence is SEQ ID NO: 76 or sequence variant thereof; the variable light chain CDR2 sequence is SEQ ID NO: 75 or sequence variant thereof; and the variable light chain CDR1 sequence is SEQ ID NO: 74 or sequence variant thereof, and wherein the sequence variant comprises one, two or three amino acid substitutions in the cited sequence. binds to a human c-Met receptor protein, the antibody or antigen binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the variable heavy chain domain comprises a VH sequence with at least 85 % sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, for the amino acid sequence shown as: SEQ ID NO: 77.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises the VH amino acid sequence shown as: SEQ ID NO: 77.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain The lightweight variable comprises a V Lambda sequence with at least 80% of at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity, for the amino acid sequence shown as SEQ ID NO: 78.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain light variable comprises the amino acid sequence V Lambda shown as SEQ ID NO: 78.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises a VH sequence with at least 85% of the sequence identity, or at least 90% of the sequence identity, or at least 95% of the sequence identity, or at least 97%, 98% or 99% of the sequence identity sequence, for the amino acid sequence shown as: SEQ ID NO: 77; and the variable light chain domain comprises a V Lambda sequence with at least 80% of the sequence identity, or sequence identity, or at least 97%, 98% or 99% of the sequence identity, for the amino acid sequence shown as SEQ ID NO: 78.
An antibody or antigen-binding region that binds to a human c-Met receptor protein, the antibody or antigen-binding region comprising a variable heavy chain domain and a variable light chain domain, wherein the chain domain heavy variable comprises the VH amino acid sequence shown as: SEQ ID NO: 77; and the variable light chain domain comprises the V Lambda sequence with at least 80% of the sequence identity, or at least 85% of the sequence identity, or at least the amino acid sequence shown as SEQ ID NO: 78. Calculation of% of sequence identity
Unless otherwise indicated in the present patent application, the% of the sequence identity between two amino acid sequences can be determined by comparing these two sequences ideally aligned and in which the amino acid sequence to be compared may comprise additions or deletions. with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated The amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100 in order to obtain the percentage of identity of these two sequences. For example, it is possible to use the BLAST program, "BLAST 2 sequences" (Tatusova et al, "Blast 2 sequences a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247250) available at http: // www.ncbi.nlm.nih.gov/ gorf / bl2.html, the parameters used here are those given by default (in particular for the parameters like "open gap penalty": 5, and "extension gap penalty": 2; a chosen matrix being, for example, the "BLOSUM 62" matrix proposed by the program), the percentage of identity between the two sequences to be compared calculated directly by the program.
Combinations of antibodies / antigen-binding regions
Exemplary, but not limiting combinations of antibodies or antigen-binding regions for inclusion in the production of the product combination or composition, or the multivalent antibody provided here are as follows: 48A2 and variants thereof combined with 36C4 and 4 8A2 and variants thereof combined with 13E6 and variants thereof; 3604 and variants thereof combined with 20F1 and variants thereof; and 36C4 and variants thereof combined with 34H7 and variants thereof.
The combination of 48A2 and variants thereof with 36C4 and variants thereof is particularly preferred, both as a combination or composition of the product and a multi-specific antibody.
References here to "combination of 48A2 and variants thereof with 36C4 and variants thereof" should be taken to end combinations formed from any variants of 48A2 and competing antibodies described above combined with any of the variants of 36C4 and competing antibodies described above.
In one embodiment the combination or composition of the product can comprise a first antibody that binds to an epitope within the 523 peptide sequence a second antibody that binds to an epitope within the 98-VDTYYDDQLISCGSVNRGTCQRHVFPHNHTAD IQSEVHCIFSPQIEEPSQCPDCWSALGAKVLSSVKDRFINFFVGNTINSSYFP DHPLHSISVRRLKETK-181 (NO1) domain name in the # 4 EMC Domain # 181 (SEQ ID NO: 36)
The multi-specific antibody may comprise a first antigen binding region that binds to an epitope within the peptide 523-RVERCLSGTWTQQICLPAIYKVFPNSA PLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVG PAMNKHFNMSIIISNGHGTTQYSTFSYVDP-633 (SEQ ID NO: 13 6) in the PSI-IPT1 region of human c-Met protein and a second antigen-binding region that binds to an epitope within the 98-VDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCWS ALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRQKK-4 domain name] (ID-184) NO human domain: 36
The combination of 4 8A2 and 36C4 is particularly advantageous because it exhibits very potent inhibition of HGF-independent c-Met activation, and also antagonizes HGF-dependent activation of the c-Met receptor, while also exhibiting an extremely low level of agonist activity. .
The combination of 48A2 and 36C4 is also particularly advantageous because it can block both HGF binding to the high affinity HGF binding site in the low affinity HGF binding in human c-Met.
Other properties of the product or multi-specific antibody combination or composition
The product combination or composition, or the multi-specific antibody, provided here may each exhibit one or more, or any combination, of the following properties / characteristics:
The product combination or composition, or the multi-specific antibody, acts as an inhibitor of HGF independent activation of the human c-Met receptor.
The product combination or composition, or the multi-specific antibody, can inhibit HGF-independent dimerization, and more particularly homodimerization and / or heterodimerization, of the human c-Met protein.
The product combination or composition, or the multi-specific antibody, acts as a strict antagonist of the HGF-mediated activation of the human c-Met receptor.
The individual antibodies present in the product combination or composition, or the multi-specific antibody, may exhibit one or more effector functions selected from antibody dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and cell-mediated phatosis dependent on
Human met on the cell surface.
The individual antibodies present in the product combination or composition, or the multi-specific antibody, can exhibit ADCC against cancer cells addicted to cMet.
The individual antibodies present in the product combination or composition, or the multi-specific antibody, can exhibit increased ADCC function in comparison to the reference antibody which is an equivalent antibody comprising a native human Fc domain. In a non-limiting embodiment, the ADCC function can be increased by at least 10x compared to the reference antibody comprising a native human Fc domain. In this "equivalent" context it can be interpreted as meaning that the antibody with increased ADCC function exhibits substantially identical antigen binding specificity and / or shares an identical amino acid sequence with the reference antibody, except for any modifications made (relating to human Fc native) for the purposes of increasing ADCC.
The individual antibodies present in the product combination or composition, or the multi-specific antibody may contain a hinge region, CH2 and CH3 domain of IgG
The individual antibodies present in the product combination or composition, or the multi-specific antibody can include modifications in the Fc region, as explained elsewhere here. In particular, the individual antibodies present in the product combination or composition, or the multi-specific antibody, can be a non-fucosylated IgG.
In other respects, the invention also provides polynucleotide molecules that encode the individual antibodies present in the product combination or composition, or that encode components of the multi-specific antibody (i.e., individual heavy or light chains of the same), in addition to vectors of expression comprising polynucleotides, host cells containing the vectors and methods of expression / recombinant production of c-Met antibodies.
In yet another aspect, the combination or composition of the product can be provided as a pharmaceutical composition designed for human therapeutic use.
The invention further provides a pharmaceutical composition comprising the multi-specific antibody described herein and a pharmaceutically acceptable carrier or excipient.
In yet another aspect, the invention relates to medical treatment methods using a combination or composition of cancer treatment, including both HGF-dependent cancers and HGF-independent cancers. Definitions
"Product combination or composition" - As used herein, the term "product combination or composition" refers to any product or composition containing two or more antibodies, or antigen-binding fragments thereof, each of which binds to a human c-Met receptor protein. A "composition" can be formed by simply mixing two or more c-Met component antibodies. The relative proportions of the two or more component c-Met antibodies within the mixture may vary. In the case of a composition comprising two c-Met antibodies, the component antibodies can be present in an approximate 1: 1 mixture. The term "composition" may include compositions designed for therapeutic use. The term "product combination" may encompass products of the combination in which two or more component antibodies are packaged within a single product or article of manufacture, but are not necessarily mixed in the mixture. "Antibody" or "Immunoglobulin" -As used herein, the term "immunoglobulin" includes a polypeptide having one or not having any relevant specific immunoreactivity. "Antibodies" refer to such pools that have specific known immunoreactivity activity significant for an antigen of interest (for example, human cMet). The term "c-Met antibodies" is used here to refer to antibodies that exhibit immunological specificity for human c-Met protein. As explained elsewhere here, "specificity" for human c-Met does not exclude cross-reaction with homologous species of c-Met. Antibodies and immunoglobulins comprise light and heavy chains, with or without an inter-chain covalent bond between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.
The generic term "immunoglobulin" comprises five distinct classes of antibodies that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight of approximately 23,000 Daltons, and two identical heavy chains of molecular weight of 53,000 to 70,000. The four chains are connected by links r) 1 ociil f obn iimo r'nnf i li V H «m rrma o o" Ipvoα supports heavy chains starting at the mouth of the "Y" and continuing through the variable region.
The light chains of an antibody are classified as either kappa or lambda (K, A,). Each heavy chain class can be linked with either a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when immunoglobulins are generated by both hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequence passes from an N at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, p, α, δ, ε) with some subclasses between them (for example, Ü1-DD4). It is the nature of this chain that determines the "class" of the antibody such as IgG, IgM, IgA IgG, or IgE, respectively. The subclasses of immunoglobulins (isotypes), for example, IgGl, IgG2, IgG3, IgG4, IgAl, etc. they are well characterized and are known to confer functional specialization.
Modified versions of each of these classes and isotypes are readily discernible by those skilled in the art in view of the present description and, consequently, are within the scope of the present invention.
As indicated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary structure of the antibody forms the antigen binding site present at the end of each Y arm. More specifically, the antigen binding site is defined by three complementary determinant regions (CDRs) on each of the VH and VL chains.
"C-Met protein" or "c-Met receptor" As used herein, the terms "c-Met protein" or "c-Met receptor" or "c-Met" are used interchangeably and refer to the receptor tyrosine kinase which, in its wild type, binds to the Hepatocyte Development Factor (HGF). The terms "human c-Met protein" or "human cMet receptor" or "human c-Met" are used interchangeably to refer to human c-Met, including the human c-Met protein naturally expressed in the human host, as well as as recombinant forms and fragments thereof and also naturally occurring mutant forms, polymorphic variants and functionally active mutant forms. Specific examples of the human c-Met include, for example, the human peptide encoded by the nucleotide sequence provided in GenBank Acc No. NM_000245, or the human protein encoded by the polypeptide sequence provided in GenBank Acc. No. NP_000236, or their extracellular domain. The single chain precursor c-Met protein is cleaved after translation to produce the alpha and beta subunits, which are disulfide linked to form the mature receptor. The cMet antibodies provided here typically bind both the mature human c-Met protein and expressed on the cell surface, for example, as expressed in the MKN-45 gastric cell line as the recombinant human c-Met protein (for example, c-Met recombinant dimeric obtained in R&D systems, 358-MT / CF).
"Binding Site" As used herein, the term "binding site" comprises a region of a polypeptide that is responsible for selective binding to a target antigen of interest (e.g., human c-Met). Binding domains or binding regions comprise at least one binding site. Exemplary binding domains include a variable antibody domain. The antibody molecules described herein can comprise a single or multiple antigen binding site (e.g., two, three or four) antigen binding sites.
"Derived From" As used herein the term "derived from" a designated protein (for example, a c-Met antibody or antigen-binding fragment thereof) refers to the origin of the polypeptide. In one embodiment, the polypeptide or sequence of amino acids that are derived from a particular starting polypeptide is a CDR sequence or sequence related thereto. In one embodiment, the amino acid sequence that is derived from a particular starting polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from a starting antibody. In one embodiment, the polypeptide or amino acid sequence that is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof that the portion consists of of at least 3 to 5 amino acids, 5 to 10 amino acids, at least 10 to 20 amino acids, at least 20 to 30 amino acids, or at least 30 to 50 amino acids, or that is otherwise identifiable. its origin in the starting sequence. In one embodiment, the one or more CDR sequences derived from the starting antibody are altered to produce variant CDR sequences, for example, affinity variants, wherein the variant CDR sequences maintain c-Met binding activity.
"Camelid-Derivative" In certain preferred embodiments, the cMet antibody molecules described here may comprise the structure of amino acid sequences and / or CDR amino acid sequences derived from a conventional camelid antibody that arose by active immunization of a camelid with a c-Met antigen. However, c-Met antibodies comprising amino acid sequences derived from camelids can be engineered to understand the structure and / or sequences of the constant region derived from a human amino acid sequence or from other non-camelid mammal species. For example, a human or non-human primate framework region, heavy chain portion, and / or hinge portion may be included in the object c-Met antibodies. In one embodiment, one or more non-camelid amino acids may be present in the framework region of a "camelid-derived" c-Met antibody, for example, a sequence of amino acids with camelid framework may comprise one or more amino acid mutations wherein the corresponding human or non-human primate amino acid residue is present. In addition, camelid-derived VH and VL domains, or humanized (or germinalized) variants thereof, can be linked to the constant domains of human antibodies to produce a chimeric molecule, as extensively described elsewhere here.
"Conservative amino acid substitution" - A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (for example, example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched beta side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, fani 1 al ani na tvi nf-nfann h-iRtirfina) DASAA mnrin. nm non-essential amino acid residue in an immunoglobulin polypeptide can be replaced with another amino acid residue from the same side chain family. In another embodiment, a strand of amino acids can be replaced with a structurally similar strand that differs in order and / or composition from members of the side chain family.
"Heavy Chain Portion" As used herein, the term "heavy chain portion" includes amino acid sequences derived from the constant domains of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CHI domain, a hinge domain (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, a binding molecule of the invention can comprise the Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, a binding molecule of the invention does not have at least a portion of a constant domain (for example, all or part of a CH2 domain). In certain embodiments, at least one, and preferably all, human immunoglobulins. For example, in a preferred embodiment, the heavy chain portion comprises a complete human hinge domain. In other preferred embodiments, the heavy chain portion comprising a complete human Fc portion (e.g., hinge domain, CH2 and CH3 sequences of a human immunoglobulin). In certain embodiments, the constant domains that make up the heavy chain portion are different immunoglobulin molecules. For example, a portion of a polypeptide heavy chain may comprise a CH2 domain derived from an IgGl molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are chimeric domains comprising portions of different immunoglobulin molecules. For example, a hinge can comprise a first portion of an IgGl molecule and a second portion of an IgG3 or IgG4 molecule. As reported above, it will be understood by those skilled in the art that the domains contained in the heavy chain portion can be modified so that they vary in the naturally occurring amino acid sequence of the immunoglobulin molecule (wild type). That is, the polypeptides of the invention described herein can comprise changes or modifications to one or more of the heavy chain constant domains (CHI, hinge, CH2 or CH3) and / or to the light chain constant domain (CL). Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.
"Chimeric" A "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. Amino acid sequences can normally exist in separate proteins that are joined in the fusion polypeptide, or they can normally exist in the same protein, but are placed in a new arrangement in the fused polypeptide. A chimeric protein can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the regions of the peptide are encoded in the desired relationship. Exemplary chimeric c-Met antibodies include fusion proteins comprising camelid-derived VH and VL domains, or humanized (or germinalized) variants thereof, fused to the domains contained in a human antibody, for example, IgGl, IgG2, IgG3 or Human IgG4.
"Variable region" or "variable domain" The term "variable" refers to the fact that certain portions of the VH and VL variable domains differ extensively in the sequence between antibodies and are used in the binding and specificity of each particular antibody to its target antigen . However, the variability is not evenly distributed across the variable domains of the antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains that form part of the antigen binding site. The first, second and third variable loops of the V Lambda light chain domain are referred to here as L1 (À), L2 (À) and L3 (À) and can be defined as comprising residues 24-33 (L1 (À), consisting of of 9, 10 or 11 amino acid residues), 49-53 (L2 (À), consisting of 3 residues) and 90-96 (L3 (À), consisting of 5 residues) in the VL domain (Morea et al., Methods 20: 267-279 (2000)). The first, second and third hypervariable loops of the V Kappa light chain domain are referred to here as L1 (K), L2 (K) and L3 (K) and can be defined as comprising residues 25-33 (L1 (K), consisting of of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2 (K), consisting of 3 residues) and 90-97 (L3 (K), consisting of 6 residues) in the VL domain (Morea et al., Methods 20: 267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to here as Hl, H2 and H3 and can be defined as comprising residues 25-33 (Hl, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of of 3 or 4 residues) and 91105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20: 267-279 (2000)).
Unless otherwise indicated, the terms LI, L2 and L3 refer to the first, second and third hypervariable loops of a VL domain, respectively, and contain hypervariable loops obtained from both Vkappa and Vlambda isotypes. The terms H1, H2 and H3 refer to the first, second and third hypervariable loops of the VH domain, respectively, and contain hypervariable loops obtained from any of the known chain isotypes, including yf z, δ, ot or p.
The hypervariable loops Ll, L2, L3, Hl, H2 and H3 can each comprise part of a "complementary determining region" or "CDR", as defined below. The terms "hypervariable loop" and "complementary determinant region" are not strictly synonymous, since hypervariable loops (HVs) are defined based on the structure, while complementary determinant regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983) and the limits of HVs and CDRs may be different in some VH and VL domains.
The CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the variable light chain domain, and residues 31-35 or 31-35b (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the variable heavy chain domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, ND. (1991)). Thus, HVs can be included within the corresponding CDRs and references here to the "hypervariable ties" of the VH and VL domains should be interpreted as also enclosing the corresponding CDRs, and vice versa, unless otherwise indicated.
The most highly conserved portions of the variable domains are called the framework region (FR), as defined below. The native domains of native heavy and light chains comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a β-leaf configuration, connected by the three hypervariable loops. The hypervariable bonds in each chain are kept together in close proximity by the FRs and, with the hypervariable bonds in the other chain, contribute to the formation of the anti-chlorine antigenic binding site. The structural analysis of the antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontane et al., J. Mol. Biol, 215: 175-182 (1990)). Despite their high variability in the sequence, five of the six loops adopt only a small repertoire of main chain conformations, called "canonical structures". These conformations are, firstly determined by the length of the loops and secondly by the presence of key residues in certain positions of the loops and regions of the framework that determine the conformation through their packaging, hydrogen bonding or the ability to assume non-configurations. main chain.
"CDR" —As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen combination sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Rabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) where definitions include overlaps or subsets of amino acid residues when compared to each other. The amino acid residues that contain the CDRs as defined by each of the references cited above are reported for comparison. Preferably, the term
"CDR" is a CDR as defined by Kabat based on sequence comparisons, open gap penalty ": Table 1: CDR definitions
3The residue numbering follows the nomenclature of Kabat et al., Supra 2The residue numbering follows the nomenclature of Chothia et al., Supra 3The residue numbering follows the nomenclature of MacCallum et al., Supra "Region of the framework" The term " framework region "or" FR region "as used here, includes residues and amino acids that are part of the variable region, but are not part of the CDRs (for example, using the Kabat definition of the CDRs). For this reason, a variable region framework is between about 100 to 120 amino acids in length, but includes only those amino acids outside the CDRs. For the specific example of a variable heavy chain region and for CDRs as defined by Kabat et al., The region of framework 1 corresponds to the domain of the variable region containing amino acids 1-30; the region of framework 2 corresponds to the domain of the variable region containing amino acids 36-49; the region of framework 3 corresponds to the domain of the variable region enclosing amino acids 66-94, and the region of framework 4 corresponds to the domain of the variable region enclosing amino acids 103 for the end of the variable region. The regions of the framework for the light chain are similarly separated by each of the CDRs of the variable light chain region. Similarly, using the definition of CDRs by Chothia et al. or McCallum et al. the boundaries of the framework region are separated by the end of the respective CDR as described above. In preferred embodiments the CDRs are as defined by Kabat.
In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen-binding site as the antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the heavy and light variable domains show less intermolecular variability in the amino acid sequence and are called regions of the framework. The regions of the framework largely adopt a βsheet conformation and the CDRs form bonds that connect, and in some cases form part of the β-sheet structure. In this way, these regions of the framework act to form a framework that provides the positioning of the six CDRs in the correct orientation by inter-chain interactions, not covalent. The antigen binding site formed by the positioned CDRs defines a complementary surface to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the epitope of the immunoreactive antigen. The position of the CDRs can be quickly identified by those skilled in the art.
"Hinge region" As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al.J. Immunol. 1998 161: 4083). C-Met antibodies comprising a "completely human" hinge region can contain one of the hinge region sequences shown in Table 25 below. Table 2: human hinge sequences

"CH2 domain" As used herein the term "CH2 domain" includes the portion of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360 , Kabat numbering system, and residues 231-340, EU numbering system, Kabat EA et al. Sequences of Proteins of Immunological Interest (Bethesda, US Department of Health and Human Services, NIH 1991). The CH2 domain is unique because it is not closely matched to another domain. Instead, the two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.
"Fragment" The term "fragment" refers to a part or portion of an antibody or antibody chain comprising less amino acid residues than an intact or complete antibody or antibody chain. The term "antigen binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to the antigen or competes with the intact antibody (i.e., with the intact antibody from which they were derived) for binding to the antigen (ie, specific binding to human c-Met). As used herein, the term "fragment" of an antibody molecule includes antigen-binding fragments of antibodies, for example, an antibody variable light chain (VL) domain, an antibody variable heavy chain domain (VH), a single chain antibody (scFv), an F (ab ') 2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a single domain antibody (DAb) fragment, an antibody with a (monovalent) arm, or any antigen binding molecule formed by combining, assembling or conjugating such antigen binding fragments. Fragments can be obtained, for example, via the chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.
"Valency" As used here the term "valence" refers to the number of potential target binding sites on a polypeptide. Each target binding site specifically binds a target molecule or specific site on a target molecule. When a polypeptide comprises more than one antigen binding site, each target binding site can specifically bind to the same or different molecules (for example, it can bind to different ligands or antigens, or different epitopes on the same antigen). The individual antibodies present as components of a combination or composition of the product preferably have at least one specific binding site for a human c-Met molecule, while the multi-specific antibodies provided here have by definition at least two different binding sites for the human c-Met, having different binding specificities. In particular embodiments the c-Met antibodies provided herein as components of the combination or composition
"Specificity" The term "specificity" refers to the ability to bind (for example, immunoreact with) a given target, for example, c-Met. A polypeptide can be monospecific and contain one or more binding sites that specifically bind to a target, or a polypeptide can be multi-specific and contain two or more binding sites that specifically bind to the same or different targets. The individual antibodies present as components of a combination or product composition can be specific to more than one target, for example, they can bind to c-Met and a second molecule expressed in a tumor cell. In another embodiment, the multi-specific antibody of the invention that binds to two or more binding sites on human c-Met may have another binding specificity, for example, for a second molecule expressed in a tumor cell. Exemplary antibodies that comprise antigen binding sites that bind antigens expressed on tumor cells are known in the art and one or more CDRs of such antibodies can be included in the antibody of the invention.
"Synthetic" As used here, the term "synthetic" with respect to polypeptides includes polypeptides that comprise a sequence of amino acids that is not naturally occurring. For example, non-naturally occurring polypeptides that are modified forms of naturally occurring polypeptides (for example, comprising a mutation such as an addition, substitution or deletion) or that comprise a first sequence of amino acids (which may or may not be occurring naturally) that it is linked in a linear sequence of amino acids with a second sequence of amino acids (which may or may not be occurring naturally) with which it is not naturally linked in nature.
"Engineered" As used herein, the term "engineered" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (for example, by recombinant techniques, peptide synthesis in vitro, by enzymatic or chemical copulation of the peptides or some combination of these techniques ). Preferably, the antibodies of the invention are engineered, including, for example, humanized and / or chimeric antibodies, and antibodies that have been engineered to improve one or more properties, such as antigen binding, stability / half-life or effector function. '' Modified antibody "As used here, the term rip>. <=: I nt Áti na.q" ant i nnmn mnr] ifi raHn "1nrlui fnrma« antibodies that are altered so that they are not naturally occurring, for example , antibodies comprising at least two heavy chain portions, but not two complete heavy chains (such as antibodies or minibodies deleted from the domain); multispecific forms of antibodies (e.g., bi-specific, triespecific, etc.) altered to bind to two or more different antigens or different epitopes on a single antigen), heavy chain molecules linked to SscFv molecules and the like ScFv molecules are known in the art and are described, for example, in US patent 5,892,019. , the term "modified antibody" includes multivalent forms of antibodies (e.g., tri-valent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen). In another embodiment, a modified antibody of the inventionis a fusion protein comprising at least a heavy chain portion without a CH2 domain comprising a polypeptide binding domain comprising the binding portion of a member of a receptor ligand pair.
The term "modified antibody" can also be used here to refer to the amino acid sequence variants of a c-Met antibody. It will be understood by those skilled in the art that a c-Met antibody code be modified to produce a variant of the c-Met antibody that varies in amino acid sequence compared to the c-Met antibody from which it was derived. For example, substitutions of nucleotides or amino acids leading to conservative substitutions or changes in "non-essential" amino acid residues can be made (for example, in CDR and / or framework residues). Amino acid substitutions may include replacing one or more amino acids with a naturally occurring or unnatural amino acid.
"Humanized Substitutions" As used herein, the term "humanized substitutions" refers to amino acid substitutions where the amino acid residue present at a particular position in the VH or VL domain of the antibody of the c-Met antibody (for example, a c -Met derived from camelid) is replaced with an amino acid residue that occurs in an equivalent position in a reference human VH or VL domain. The reference human VH or VL domain can be a VH or VL domain encoded by the human germline, in which case the substituted residues can be referred to as "germinal substitutions". Humanized / germinal substitutions can be made in the framework regions and / or in the CDRs of a c-Met antibody, defined here. "Affinity Variants" As used here, the term "affinity variant" refers to an antibody variant that exhibits one or more changes in the amino acid sequence compared to a reference c-Met antibody, where the affinity variant exhibits a altered affinity for human c-Met protein compared to the reference antibody. Typically, affinity variants will exhibit improved affinity for human c-Met, as compared to the reference c-Met antibody. The improvement can be either a lower KD, for human c-Met, or a faster shutdown rate for human c-Met or a change in the pattern of cross-reactivity with non-human c-Met counterparts. Affinity variants typically exhibit one or more changes in the amino acid sequence in the CDRs, as compared to the reference c-Met antibody. Such substitutions may result in the substitution of the original amino acid present at a given position in the CDRs with a different amino acid residue, which may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. Amino acid substitutions can be conservative or non-conservative.
"High human homology" An antibody comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain will be considered to have high human homology if the VH and VL domains, taken together, exhibit at least 90 % of the amino acid sequence identity for the most germinal human germline VH and VL sequences. Antibodies having high human homology can include antibodies comprising VH and VL domains of native non-human antibodies that exhibit a sufficiently high% of the sequence identity of human germline sequences, including, for example, antibodies comprising VH and VL domains of conventional antibodies of camelids, as well as engineered, especially humanized, variants of such antibodies and also "completely human" antibodies.
In one embodiment, the VH domain of the antibody with a high human homology can exhibit an amino acid sequence identity or sequence homology of 80% or greater with one or more human VH domains across the FR1, FR2, FR3 and FR4. In other embodiments, the identity of the amino acid sequence or sequence homology between the VH domain of the polypeptide of the invention and the closest matching human germline VH domain sequence can be 85% or greater, 90% or greater, 95 % or greater, 97% or greater, or up to 99%
In one embodiment, the VH domain of the antibody with high human homology may contain one or more (for example, 1 to 10) mismatched amino acid sequences across the FR1, FR2, FR3 and FR4 framework region, compared to closest matching human VH sequence.
In another embodiment, the VL domain with high human homology may exhibit a sequence identity or sequence homology of 80% or greater with one or more human VL domains across the FR1, FR2, FR3 and FR 4 framework regions. embodiments the identity of the amino acid sequence or sequence homology between the VL domain of the polypeptide of the invention and the sequence of the closest matching human germline VL domain can be 85% or greater 90% or greater, 95% or greater , 97% or greater, or up to 99% or even 100%.
In one embodiment, the VL domain of the antibody with high human homology may contain one or more (for example, 1 to 10) mismatched amino acid sequences across the FR1, FR2, FR3 and FR4 framework regions, compared to closest matching human VL sequence.
Before analyzing the percentage of sequence identity between the antibody with high human homology and human germline VH and VL, canonical folds can be determined, which allows the identification of the family of human germline segments with the identical combination of canonical folds for Hl and H2 or LI and L2 (and L3). Subsequently the member of the human germline family that has the highest degree of sequence homology with the variable region of the antibody of interest is chosen to classify the sequence homology. The determination of the Chothia canonical classes of the hypervariable loops Ll, L2, L3, Hl and H2 can be determined with the bioinformatics tools available publicly on the website www.bioinf.org.uk/abs/chothia.html.page. Providing the program output shows the requirements for the key residue in a data file. In these data files, the positions of the key residues are shown with the amino acids allowed in each position. The sequence of the variable region of the antibody of interest is given as an entry and is first aligned with a consensus antibody sequence to designate the Kabat numbering scheme. The analysis of the canonical folds uses a standard set of key residues derived from an anf-nmat-i 7sr) n method Hpsprivnl vi dn r> r> T Martin A Thnrntnn (Martin A / al., J. Mol. Biol. 263: 800-815 (1996)).
With the V segment of a particular known human germline that uses the same combination of canonical folds for H1 and H2 or LI and L2 (and L3), the member of the family that best matches in terms of sequence homology can be determined. With bioinformatics tools the percentage of sequence identity between the amino acid sequences of the VH and VL domain framework of the antibody of interest and corresponding sequences encoded by the human germline can be determined, but the actual manual alignment of the sequences can also be applied . Human immunoglobulin sequences can be identified from various databases, such as VBase (http://vbase.mrccpe.cam.ac.uk/) or the Pluckthun / Honegger database (http: //www.bioc .unizh.ch / antibody / Sequences / Germlines To compare human sequences with the V regions of the VH or VL domains in an antibody of interest, a sequence alignment algorithm as available via websites like www.expasy.ch/tools / # align can be used, but manual alignment with the limited set of sequences can be performed.The light and heavy chain sequences of human germline from families with high homology to skeleton regions 1, 2, and 3 from each chain are selected and compared with the variable region of interest, the FR4 is also checked against the JH and JK or JL regions in the human germ line.
Note that in calculating the total percentage of the sequence homology, the FR1, FR2 and FR3 residues are evaluated using the combination sequence closest to the human germline family with the identical combination of canonical folds. Only residues other than members of the closest combination or others of the same family with the same canonical fold combination are classified (NB excluding any first coded differences). However, for humanization purposes, residues in the skeleton regions identical to members of other families of human germline, which do not have the same combination of canonical folds, can be considered "human", despite the fact that these can be classified as "negative" under the strict conditions described above. This assumption is based on the "mix and match" approach for humanization, where each of FR1, FR2, FR3 and FR4 is compared separately with its closest matching germline sequence and the humanized molecule therefore contains a combination different from FRs as was done by Qu and colleagues (Qu et la., Clin.
Cancer Res. 5: 3095-3100 (1999)) and Ono and colleagues (Ono et al., Mol. Immunol. 36: 387-395 (1999)). The borders of the individual skeleton regions can be accessed using the IMGT numbering scheme, which is an adaptation of the Chothia numbering scheme (Lefranc et al., NAR 27: 209-212 (1999); http: //imgt.cines .fr).
Antibodies with high human homology may comprise hypervariable loops or CDRs having human or human-like canonical folds, as discussed in detail below.
In one embodiment at least one hypervariable loop or CDR in both the VH and VL domain of the antibody with high human homology can be obtained or derived from a VH or VL domain of a non-human antibody, for example, a conventional antibody from a Camelidae species, still exhibits a predicted or actual canonical fold structure that is identical to the canonical fold structure that occurs in human antibodies.
It is well established in the art that, although the primary amino acid sequences of the hypervariable loops present in both the VH and VL domains encoded by the human germline are, by definition, highly variable, all hypervariable loops, except the CDR H3 rio dnmín-irt pineapples distinct structural, called canonical folds (Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Tramontane et al. Proteins 6: 382-94 (1989)), which depend both the length of the hypervariable loop and the presence of the so-called canonical amino acid residues (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)). The actual canonical structures of the hypervariable loops in the intact VH or VL domains can be determined by structural analysis (for example, X-ray crystallography), but it is also possible to predict the canonical structure based on the key amino acid residues that are characteristic of a structure (discussed further below). In essence, the specific pattern of residues that determine each canonical structure forms a "signature" that allows the canonical structure to be recognized in the hypervariable loops of a VH or VL domain of the unknown structure; canonical structures can therefore be predicted based on the amino acid sequence alone.
The canonical fold structures predicted for the hypervariable loops of any given VH or VL sequence in an antibody with high human homology can be analyzed using algorithms that are publicly available at www.biochem.ucl.ac.uk/~martin/anticorpos.html and www .bioc.unizh.ch / antibody / Sequences / Germlines / Vbase_hVk.h tml. These tools allow to question the VH or VL sequences to be aligned against the sequences of the human VH or VL domain of known canonical structure, and a prognosis of the canonical structure made for the hypervariable ties of the questioned sequence.
In the case of the VH domain, the H1 and H2 loops can be classified as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if at least the first, preferably both, of the following criteria are satisfied: 1. An identical length to, determined by the number of residues, for the closest canonical human structural class of combination. 2. At least 33% of identity, preferably at least 50% of identity with the key amino acid residues described for the corresponding human canonical classes H1 and H2. (Note that for the purposes of the preceding analysis the Hl and H2 loops are treated separately and compared against their human canonical structural class of most closely matching)
The preceding analysis relies on the prognosis of the canonical structure of the H1 and H2 loops of the antibody of interest. If the actual structures of the Hl and H2 loops in the antibody of interest are known, for example, based on X-ray crystallography, then the Hl and H2 loops in the antibody of interest can also be classified as having a canonical fold structure " substantially identical "to a canonical fold structure known to occur in human antibodies if the loop length differs from that of the closest combination human canonical structural class (typically ± 1 or +2 amino acid), but the actual structure of the H1 and H2 in the antibody of interest matches the structure of a human canonical fold.
Key residues and amino acids found in the canonical structural classes for the first and second hypervariable loop of the human VH domains (H1 and H2) are described by Chothia et al., J. Mol. Biol. 227: 799-817 (1992), the contents of which are incorporated here in their entirety as a reference. In particular, Table 3 on page 802 and Chothia et al., Which is specifically incorporated here as a reference, lists the preferred amino acid residues at key sites for the canonical Hl structures found in the human germ line, while Table 4 on page 803 , also incorporated specifically as a reference, lists the preferred amino acid residues at key sites for the canonical structures of the CDR H2 found in human germline.
In one embodiment, both H1 and H2 in the VH domain of the antibody with high human homology exhibit a predicted or actual canonical fold structure that is substantially identical to a canonical fold structure that occurs in human antibodies.
Antibodies with high human homology can comprise a VH domain in which the hypervariable loops H1 and H2 form a combination of canonical fold structures that are identical to a combination of canonical structures known to occur in at least one VH domain of the human germline. It has been observed that only certain canonical fold structures in Hl and H2 actually occur in VH domains encoded by the human germ line. In an H1 and H2 embodiment in the VH domain of the antibody with high human homology, they can be obtained from a VH domain of a non-human species, for example, a Camelidae species, yet form a combination of predicted canonical fold structures or which is identical to a combination of canonical fold structures known to occur in a human germline or VH domain modified by somatically mutation. In non-limiting embodiments H1 and H2 in the VH domain of the antibody with high human homology, they can be obtained from a VH domain of a non-human species, for example, a Camelidae species, and form one of the following canonical fold combinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5.
An antibody with high human homology can contain a VH domain that exhibits both sequence identity / high sequence homology to human VH, and that contains hypervariable loops exhibiting structural homology to human VH.
It can be advantageous for the canonical folds present in Hl and H2 in the VH domain of the antibody with high human homology, and the combination of them, being "correct" for the human VH germline sequence that represents the closest combination with the VH domain of the antibody with high human homology in terms of the identity of the total primary amino acid sequence. As an example, if the combination of the closest sequence is with a human germline VH3, then it can be advantageous for H1 and H2 to form a combination of canonical folds that also occur naturally in a human VH3 domain. antibodies with high human homology that are derived from non-human species, for example, antibodies containing VH and VL domains that are derived from conventional camelid antibodies, especially antibodies containing VH and VL domains from humanized camelid.
Thus, in one embodiment the VH domain of a high human homology c-Met antibody may exhibit a sequence identity or sequence homology of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VH domain across the FR1, FR2, FR3 and FR4 framework regions, and in addition, Hl and H2 in the same antibody are obtained from a non-human VH domain (for example, derived from Camelidae species), but form a combination of the predicted or actual canonical fold structures that is the same as a canonical combination known to occur naturally in the same human VH domain.
In other embodiments, LI and L2 in the VL domain of the antibody with high human homology are each obtained from a VL domain of a non-human species (for example, a camelid-derived VL domain), and each exhibit a structure of the predicted or actual canonical fold that is substantially identical to a structure of mm or bn finrnriO hnmorr> α
As with the VH domains, the hypervariable loops of the VL domains of both types VLambda and VKappa can adopt a limited number of canonical conformations or structures, determined in part by the length and the presence of key amino acid residues in certain canonical positions.
Within an antibody of interest having high human homology, the Ll, L2 and L3 loops obtained from a VL domain of a non-human species, for example, a Camelidae species, can be classified as having a substantially canonical fold structure " identical "to a canonical fold structure known to occur in human antibodies if at least the first, and preferably both, of the following criteria are met: 1. An identical length, determined by the number of residues, for the most appropriate human structural class of combination next. 2. At least 33% of identity, preferably at least 50% of identity with the key amino acid residues described for the corresponding human canonical L1 or L2 structural classes, both from the VLambda and VKappa repertoires. the loops LI and L2 are treated separately and each compared against its closest canonical human structural class)
The preceding analysis relies on the prognosis of the canonical structure of the Ll, L2 and L3 loops in the VL domain of the antibody of interest. If the actual structure of the Ll, L2 and L3 loops is known, for example, based on X-ray crystallography, then the Ll, L2 or L3 loops derived from the antibody of interest can also be classified as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if the loop length differs from that of the closest combination human canonical structural class (typically +1 or ± 2 amino acids), but the actual structure of the Camelidae bonds matches the human canonical fold.
The key amino acid residues found in human canonical structural classes for CDRs from the human VLambda and VKappa domains are described by Morea et al. Methods, 20: 267-279 (2000) and Martin et al., J. Mol. Biol., 263: 800-815 (1996). The structural repertoire of the human VKappa domain is also described by Tomlinson et al. EMBO J. 14: 4628-4638 (1995), and that of the ITT domain W1 1 1 n □ m c α frj 7 7 (1996). The contents of all these documents are hereby incorporated by reference. Ll and L2 in the VL domain of an antibody with high human homology can form a combination of predicted or actual canonical fold structures that are identical to a combination of canonical fold structures known to occur in a VL domain of human germline. In non-limiting embodiments Ll and L2 in the VLambda domain of an antibody with high human homology (for example, an antibody containing a camelid-derived VL domain or a humanized variant thereof) can form one of the following canonical fold combinations: 11 -7, 13-7 (A, B, C), 14-7 (A, B), 12-11, 14-11 and 12-12 (as defined in Williams et al.J. Mol. Biol. 264: 220 -32 (1996) and as shown at http://www.bioc.uzh.ch/anticorpo/Sequences/Germlines/VBase_hVL.html). In non-limiting embodiments Ll and L2 in the Vkappa domain they can form one of the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (as defined in Tomlinson et al. EMBO J. 14: 4628-38 (1995) and as shown at http://www.bioc.uzh.ch/anticorpo/Sequences/Germlines/VBase_
In another embodiment, all three of LI, L2 and L3 in the VL domain of an antibody with high human homology can exhibit a substantially human structure. It is preferred that the VL domain of the antibody with high human homology exhibits both sequence identity / high sequence homology with the human VL, and also that the hypervariable bonds in the VL domain exhibit structural homology to the human VL.
In one embodiment, the VL domain of the c-Met antibody with high human homology may exhibit a sequence identity of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with the human VL domain across the FR1, FR2, FR3 and FR4 framework regions, and in addition, the hypervariable loop LI and hypervariable loop L2 can form one of predicted or actual canonical fold structures that are the same as a canonical fold combination because they occur naturally in the same human VL domain.
It is clear that it is considered that VH domains exhibiting sequence / homology identity with the elevated sequence with human VH, as well as structural homology with human VH hypervariable loops will be combined with VL domains exhibiting sequence / homology identity with σomiΔnr-i ad mm VT. hiimnnn o ♦ "zamkxom hnmnl a structural with the hypervariable bonds of human VL to provide antibodies with high human homology containing VH / VL pairs (for example, camelid-derived VH / VL pairs) with structural homology and maximum sequence for pairs of Coded human VH / VL.
"Strict antagonist" As defined here, an antibody or antigen binding region, which acts as or is capable of acting as a "strict antagonist" of the HGF-mediated activation of the c-Met receptor has the following properties: (1) it it is an antagonist of HGF-mediated activation of the c-Met receptor, and (2) it does not exhibit significant intrinsic agonist activity.
As used here, the term "antagonist of HGF-mediated activation of the c-Met receptor" refers to a molecule, such as a c-Met antibody, that is capable of inhibiting HGF-dependent c-Met activation / signaling in an appropriate test system. Effective antagonist antibodies may be able to inhibit at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80% of the maximum effect of HGF in at least one test system capable to detect HGF-dependent c-Met activation or signaling, including, for example, an HGF-dependent c-Met phosphorylation test, or an HGF-induced tumor cell proliferation test, cell survival tests, etc. A c-Met antibody provided here can also be recognized as a potent antagonist of HGF-mediated activation of the c-Met receptor if the antagonist activity obtained is at least as potent as that obtained with the reference antibody C224G11 (as described in WO 2009 / 007427), reference antibody which is a murine-human chimeric antibody of the IgGl isotype comprising a variable heavy chain domain having an amino acid sequence shown as SEQ ID NO: 43 and the variable light chain domain having the amino acid sequence shown as SEQ ID NO: 44 and a human constant region that is not modified by the hinge, i.e., that comprises the wild-type hinge region of human IgG1.
As used herein, the term "intrinsic agonist activity" of a c-Met antibody refers to the antibody's ability to activate the c-Met receptor in the absence of the HGF ligand. Intrinsic agonist activity can be tested in an appropriate test system, for example, a phosphorylation test of c-Met in the presence and absence of HGF. In one embodiment, an antibody exhibits "significant intrinsic agonist activity" if the agonist effect produced in the absence of HGF is greater than 20%, test system. Conversely, a c-Met antibody is considered to exhibit no significant intrinsic agonist activity if the agonist effect produced in the absence of HGF is less than 20%, or less than 16%, or less than 10%, or less than than 5% of the maximum effect of HGF in the same test system. As an example, the antagonist activity and intrinsic agonist activity of a c-Met antibody can be assessed by performing a cell spread test, in the presence and absence of HGF. "Strict antagonist" antibodies, that is, without significant intrinsic agonist activity, will not typically produce a detectable spreading effect in the absence of HGF, but exhibit a strong inhibition of HGF-induced spreading in the same test system. Intrinsic agonist activity can also be assessed using the phosphorylation test described in Example 9 of the present patent application. The c-Met antibody preferably exhibits less than 20% of the maximum effect of HGF in this test system.
The individual c-Met antibodies provided here are also considered to exhibit no significant intrinsic agonist activity if the agonist effect produced in the absence of HGF is equal to or less than that obtained with the reference antibody c224Gll (as described in WO 2ΩΩ9 / ΩΩ7427 ). ant-irnrnn rip rsfprÂnria o rrual is a murine-human chimeric antigen of the IgG1 isotype comprising a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 43 and the variable light chain domain having the sequence of amino acids shown as SEQ ID NO: 44 and a human constant region that is not modified by the hinge, i.e., that comprises the wild-type hinge region of human IgG1.
The combination or composition of the product may comprise isolated antibodies (which may be monoclonal antibodies) having high human homology that specifically binds to a human c-Met receptor protein, wherein the antibodies are strict antagonists of c receptor HGF-mediated activation -Met. The properties and characteristics of c-Met antibodies, and antigen binding regions, which may be included in the product combination or composition or multi-specific antibodies according to the invention will now be described in more detail.
C-Met binding and affinity
Isolated antibodies having high human homology that specifically bind to a human c-Met receptor protein will typically exhibit a binding affinity (KD) for human c-Met, and more particularly for the human c-Met domain, of about of lOnM or less, or lnM or less, or 0, lnM or less, or 10 µM or less, and may exhibit a dissociation rate for binding to the human c-Met of 10 ^ s'1 or less, or 10'4s- 1 or less. Binding affinity (KD) and dissociation rate (kOff) can be measured using standard techniques well known to those skilled in the art, such as, for example, surface plasmon resonance (BIAcore), as described in the accompanying examples.
The c-Met antibodies described here exhibit immunological specificity for binding to human c-Met, and more specifically the extracellular domain of human c-Met, but cross-reactivity with non-human homologues of c-Met is not excluded. The binding affinity displayed with non-human primate homologues of c-Met (eg, simian c-Met rhesus) is typically 1 to 10, for example, 5 to 10 times less than the affinity for c- Human Met.
Antagonist / agonist properties
As described elsewhere, the individual c-Met antibodies provided here, and also combinations of two or more of such antibodies (or antigen-binding regions derived therefrom), and the multi-specific antibody described here, may be "strict antagonists. "of HGF-mediated activation of the human c-Met receptor. according to the definition given above. The individual c-Met antibodies, and also combinations of two or more of such antibodies (or antigen-binding regions derived therefrom), and the multi-specific antibody described here, may exhibit potent antagonism of HGF-mediated c-Met activation with minimal agonist activity. This balance between high antagonist activity and minimal intrinsic agonist activity is critical for the therapeutic usefulness of c-Met antibodies, since it has been previously demonstrated (WO 2010/069765) that the loss of in vitro antagonist activity accompanies the increase in agonist activity in chimeric form of the murine monoclonal antibody 224G11 can result in significant loss of antagonist activity in vivo.
Many in vivo in vitro tests suitable for testing the antagonism of HGF-mediated c-Met activation and / or agonist activity of c-Met antibodies and combinations thereof have been described in the art and are readily available to those skilled in the art (see, for example, WO 2010/059654, WO 2009/07427, WO 2010/069765, Pacchicina et al., JBC, manuscript M110.134031, September 2010, the technical teachings of which in relation to such tests are incorporated here by reference) . Appropriate tests include, for example, spread test, wound healing test, proliferation test, c-Met phosphorylation test, branching morphogenesis test and tests based on growth inhibition / apoptosis.
Inhibition of HGF-independent c-Met activation
The product combinations and compositions and the multi-specific antibodies described here have the ability to inhibit HGF independent activation of the c-Met receptor. In vitro tests appropriate to test the HGF independent activation of the c-Met receptor are described in the accompanying examples.
In particular embodiments, product combinations or compositions, as well as multispecific antibodies, may exhibit HGF-independent c-Met receptor activation, and more specifically may exhibit c-Met HGF-independent phosphorylation in the cell line of human MKN-45 carcinoma. In particular embodiments, product combinations or compositions, as well as multi-specific antibodies, can exhibit at least 40%, or at least 50%, or at least 60%, or at least 70% or at least 8 0% inhibition of HGF-independent c-Met receptor activation. More specifically, the combination or composition of the product (and optionally the antibodies of the individual component thereof) may exhibit at least 40% of it. or at least 50%. or at least 60%, or at least 70% or at least 80% inhibition of HGF-independent c-Met auto-phosphorylation, as measured by the phosphorylation test, for example, the phosphorylation test described here performed on the lineage of human gastric cell MKN-45.
The product combination or composition (and optionally also the antibodies of the individual component thereof) or the multi-specific antibody should preferably exhibit at least the same potency as the reference antibody C224G11 and should preferably exhibit more potent inhibition of HGF independent activation. (auto-phosphorylation) of c-Met than the reference antibody C224G11, particularly when measured by the phosphorylation test on MKN-45 cells. Some of the c-Mets antibodies provided here, in particular those comprising the 36C4, 48A2 antigen-binding domains and germline-derived variants thereof, are shown to be more potent inhibitors of c-Met independent autophosphorylation than the reference antibody C224G11, while still exhibiting comparable (or better) antagonism of HGF-independent c-Met activation than the reference antibody C224G11 and lower levels of intrinsic agonist activity than the reference antibody C224G11. In addition, the combination of 36C4 mixed with 48A2 (for example, as a 1: 1 mixture) is even more potent than the antibody component tested individually. As noted elsewhere here, the reference antibody C224G11 (as described in WO 2009/007427) is a murine-human chimeric antibody of the IgGl isotype comprising a variable heavy chain domain having the amino acid sequence shown as SEQ ID NO: 181 and the variable light chain domain having the amino acid sequence shown as SEQ ID NO: 182 and a human constant region that is not modified by the hinge, that is, that comprises the wild-type hinge region of human IgG1.
The c-Met antibodies provided here also exhibit substantially more potent inhibition of HGF independent auto-phosphorylation of c-Met than the reference antibody 5D5, which shows no inhibition in this test system.
Inhibition of c-Met dimerization
The combination or composition of the product or the multi-specific antibody provided herein preferably exhibits the ability to inhibit dimerization of c-Met receptors, and more particularly the ability to inhibit homodimerization and or heterodimerization of membrane-bound c-Met receptors present in the cell surface of tumor cells. The ability to inhibit c-Met dimerization is relevant to the therapeutic utility of c-Met antibodies, since antibodies that inhibit cMet dimerization may be usable in the treatment of cancers associated with c-Met independent of HGF, moreover, for cancers activated by HGF-dependent c-Met. The heterodimerization of c-Met is discussed in Trusolino et al., Nature Reviews, Molecular Cell Biology., 2010, 11: 834-848.
Appropriate tests to test the ability of c-Met antibodies to inhibit dimerization of c-Met have been described in the art and are readily available to those skilled in the art (see, for example, WO 2009/07427 and WO 2010/069765, the technical teaching of which with respect to such tests are incorporated here by reference)
In particular embodiments, the combination or composition of the product or the multi-specific antibody may exhibit inhibition of dimerization in a "Met addicted" cell line, such as, for example, EBC-1 cells. In particular, c-Met antibodies can exhibit at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50% inhibition of c-Met dimerization (homo) dimerization in a cell line addicted to co-occur in cell lines that exhibit stable chromosomal amplification of the MET oncogene, as described in Smolen et al, PNAS, vol.103, pp2316 -2321, 2006.
Negative regulation of cMet protein expression on the cell surface
The combinations or compositions of the product or the multi-specific antibodies provided herein preferably do not induce significant negative regulation of human c-Met protein on the cell surface. The ability of a given c-Met antibody to induce down-regulation of human c-Met protein on the cell surface can be verified using flow cytometry in a cell line expressing c-Met, such as, for example, MKN -45. In one embodiment, the c-Met antibodies provided herein are considered to not induce negative regulation of human c-Met protein to the cell surface if they induce less than 20%, or less than 15%, or less than 10% or less than 5% of the negative regulation of c-Met protein in this test system. The c-Met antibodies provided here are also considered to not induce significant negative regulation of human c-Met protein on the cell surface if they induce equal to or less than the miA A TAt-nil arãn in the neactive mode of the n-protein Met of the reference antibody C224G11 described here.
The c-Met antibodies, product combinations or compositions or multi-specific antibodies that do not induce significant negative regulation of the c-Met protein on the cell surface may be particularly appropriate which benefits the effector function of the antibody, that is, ADCC, CDC, ADCP, and in particular increased effector function. C-Met antibodies that do not induce significantly negative regulation of c-Met protein on the cell surface are not internalized, and therefore can remain bound to c-Met on the cell surface for significantly longer than c- Met that are internalized. A reduced rate of internalization (or significant lack of internalization is a distinct advantage in c-Met antibodies that exhibit effector function via at least one of ADCC, CDC or ADCP. Consequently, the c-Met antibodies described here that exhibit effector function (or increased effector function) and that do not induce significant negative down-regulation of the c-Met protein on the cell surface may be particularly advantageous for certain therapeutic applications, for example, cancer treatments that benefit from the antibody's effector function. Met
The c-Met antibodies described here bind to epitopes within the extracellular domain of human c-Met and block the binding of HGF to the extracellular domain of c-Met, to varying degrees.
The ability of the c-Met antibodies provided here to block the binding of HGF to c-Met can be measured by means of a competition test. Typically, c-Met antibodies block the binding of HGF to c-Met with an IC 50 of 0.5nM or less.
The term "epitope" refers to a specific arrangement of amino acids located in a peptide or protein with which the antibody or antibody fragment binds. Epitopes often consist of a cluster of chemically active molecules on the surface such as amino acids or sugar side chains, and have three specific dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear, that is, involving ligation with a single amino acid sequence, or conformational, that is, involving ligation with two or more amino acid sequences in several regions of the antigen that may not necessarily be contiguous.
The c-Met antibodies present in the product combination or composition or the antigen-binding regions of the multi-specific antibody can bind to different epitopes (overlapping or non-overlapping) within the extracellular domain of the human c-Met protein.
Some of the c-Met antibodies present in the product combination or composition or antigen-binding regions of the multi-specific antibody can bind to epitopes within the SEMA domain of human c-Met. The SEMA domain is contained within amino acid residues 1-491 of the mature human c-Met protein (lacking the sequence signal, as shown in Figure 25) and has been recognized in the art to contain a binding site for the HGF ligand of the c-Met.
In a particular embodiment, the cMet antibody provided here can bind to an epitope within the 98VDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCWSAL GAKVLSSVKDRFINFFVGNTINSSYVPKRHHSIS1-IDRHPLISH-human ID: 1-human identification. In particular, the antibody denoted 36C4, and the germline variants and affinity variants thereof, all bind to an epitope within this peptide region of the SEMA domain. This region of the SEMA domain is significant, as it is known to contain a binding site for the HGF ligand of c-Met. Particularly advantageous are c-Met antibodies, for example, antibodies comprising the 36C4 antigen-binding regions or one of the germinalized or affinity variants thereof, which bind to this peptide epitope within the SEMA domain of c- Met and that do not induce significant negative regulation of the c-Met protein on the cell surface. Such antibodies may also exhibit one or more effector functions selected from ADCC, CDC and ADCP, or increased effector function (s).
Other c-Met antibodies present in the product combination or composition or antigen-binding regions of the multi-specific antibody can bind to epitopes within the IPT region of human c-Met. The IPT region is known to include amino acid residues 544-909 of the mature human cMet protein lacking the peptide signal. The IPT region itself is subdivided into IPT domains 1, 2, 3 and 4, as shown in Figure 25. Through epitope mapping, it was determined that several of the c-Met antibodies described here can bind to epitopes within the IPT domains 1-2 of human c-Met (IPT-1 comprises amino acid residues 544-632 of mature human c-Met; IPT-2 comprises amino acids 633-717 of mature human c-Met), while others may bind to epitope within the IPT 2-3 domains of human c-Met (IPT-2 comprises amino acid residues 633-717 of mature human c-Met; IPT3 comprises amino acid residues 718-814 of mature human c-Met), and others can bind to the epitope within the IPT 3-4 domains of c-Met (IPT-3 comprises amino acid residues 718-814 of mature human c-Met; IPT-4 comprises amino acid residues 815-909 of c- Mature human met).
IPT 3-4 domains have been identified as containing a high affinity binding site for the HGF ligand (see, for example, EP 2119448 incorporated herein as a reference), but so far no antibody capable of binding IPT 3- domains 4 and antagonizing HGF-mediated activation dof c-Met has been described. Potent c-Met antibodies strictly antagonistic to bind to IPT domains, and particularly IPT 1-2, 2-3 and 3-4 domains, or to the PSI-IPT region of human c-Met are now provided here. Crucially, these antibodies can exhibit high human homology, as defined here, and can be provided in recombinant form containing the complete human hinge region and the Fc domain, particularly the human IgGl isotype, without significant loss of antagonistic activity or increased activity agonist. Still other cMet antibodies provided here can bind to conformational epitopes as part or all of the recognition site within the IPT of the
A specific therapeutic utility can be achieved by raising c-Met antibodies to the IPT domains, as defined above, or to junctions between the IPT domains or to conformational epitopes with all or part of the recognition site within the IPT region of the human c-Met .
Other c-Met antibodies present in the product combination or composition or antigen-binding regions of the multi-specific antibody can bind to an epitope within the human c-Met region by crossing the junction between the PSI domain and the IPT 1 (PSI domain) -IPT1). The PSI domain of human c-Met crosses amino acid residues 492-543 of mature human c-Met protein (lacking the peptide signal), while the IPT 1 domain crosses residues 544-632 of mature human c-Met. In a particular embodiment, c-Met antibody may bind to an epitope within the amino acid sequence 523EECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLL GNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDP-633 (SEQ ID NO: 136) in PSI IPT1 region of human c-Met protein. In particular, the c-Met antibody denoted here 48A2, and the germinalized strain variants and 48A2 affinity variants described here, demonstrated that if 3 -i ^ r => m => nnal rlant-m npnkíHan PQT _
IPT1 of human c-Met. The binding of a c-Met antibody to an epitope within the PSI-IPT1 region, and more specifically binding to the epitope bound by the 48A2 antibody and its variants, can produce an effect both by blocking the binding of the HGF ligand from c-Met to the binding site within the IPT region as preventing the conformational change that normally accompanies the binding of HGF to c-Met.
Camelid-derived c-Met antibodies
The antibodies present in the combination or composition of the product or in the antigen-binding regions of the multi-specific antibody can comprise at least one hypervariable loop or complementarity determining region obtained from a VH domain or a VL domain of a species of the family Camelidae, such as VH and / or VL domains, or CDRs thereof, obtained by active immunization of camelids outside the strain, for example, llamas, with a human c-Met antigen.
By "hypervariable loop or complementarity determining region obtained from a VH domain or a VL domain of a species of the Camelidae family" we mean that the hypervariable loop (HV) or CDR has an amino acid sequence that is identical, or αnhαt-anrialmonf-p ídênt-ína to RpmiÂnria HA amínnár í dns dp a hypervariable loop or CDR that is encoded by an immunoglobulin gene Camelidae. In this context "immunoglobulin gene" includes germline genes, immunoglobulin genes that have undergone re-disposition, and also genes modified by somatically mutation. Thus, the amino acid sequence of the HV or CDR obtained from a VH or VL domain of a Camelidae species can be identical to the amino acid sequence of an HV or CDR present in the conventional mature Camelidae antibody. The term "obtained from" in this context implies a structural relationship, in the sense that c-Met antibody HVs or CDRs incorporate an amino acid sequence (or minor variants thereof) that was originally encoded by an immunoglobulin Camelidae gene. However, this does not necessarily imply a particular relationship in terms of the production process used to prepare the c-Met antibody.
Camelid-derived c-Met antibodies can be derived from any species of camelid, including inter alia, llama, dromedary, alpaca, vicuna, guanaco or camel.
C-Met antibodies comprising camelid-derived VH and VL domains, or CDRs thereof, are typically recombinantly expressed polypeptides, and can be chimeric polypeptides. The term "chimeric polypeptide" refers to an artificial (non-naturally occurring) polypeptide that is created by the juxtaposition of two or more peptide fragments that do not occur otherwise contiguously. Included within this definition are "species" of chimeric polypeptides created by the juxtaposition of the peptide fragments encoded by the two or more species, for example, camelid and human.
Camelid-derived CDRs can comprise one of the CDR sequences shown as SEQ ID NOs: 1-21, 71-73 or 83-85 (heavy chain CDR) or one of the CDR sequences shown as SEQ ID NOs: 22-42, 74 -76, 86, 87 or 137-148 (light chain CDRs).
In one embodiment, the complete VH domain and / or the complete VL domain can be obtained from a species of the Camelidae family. In specific embodiments the camelid-derived VH domain can comprise the amino acid sequence shown as SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 77 or 88 while the camelid-derived VL domain can comprise the amino acid sequence shown as SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 78, 89 or 149-164. The camelid-derived VH domain and / or the camelid-derived VL domain can be linked to OTH one or more amino acid substitutions, insertions or deletions are introduced into the camelid amino acid sequence. These engineered changes preferably include amino acid substitutions in relation to the camelid sequence. Such changes include "humanization" or "germinalization" in which one or more amino acid residues in a camelid-encoded VH or VL domain are replaced with equivalent residues from a homologous human encoded VH or VL domain.
Isolated camelid VH and VL domains obtained by active immunization of a camelid (e.g., llama) with a human c-Met antigen can be used as a basis for the antigen-binding peptides engineered according to the invention. Starting from VH and VL domains of intact camelids, it is possible to engineer one or more amino acid substitutions, insertions or deletions starting from the starting camelid sequence. In certain embodiments, such substitutions, insertions or deletions may be present in the framework regions of the VH domain and / or the VL domain. The purpose of these changes in the primary amino acid sequence may presumably be to reduce unfavorable properties (for example, immunogenicity in a human host (called humanization), sites of potential product heterogeneity and or instability (glycosylation, deamidation, isomerisation, etc.) or to increase other favorable properties of the molecule (for example, solubility, stability, bioavailability, etc.). In other embodiments, changes in the primary amino acid sequence can be engineered into one or more of the hypervariable loops (or CDRs) of a domain VH and / or VL Camelidae domain obtained by active immunization, such changes can be introduced in order to increase antigen binding affinity and / or specificity, or to presumably reduce unfavorable properties, for example, immunogenicity in a human host (called humanization ), potential product heterogeneity sites and / or instability (glycosylation) deamidation, isomerisation, etc.) or to increase other favorable properties of the molecule, for example, solubility, stability, bioavailability, etc.
Thus, in one embodiment, the invention provides a variant c-Met antibody that contains at least one amino acid substitution in at least one region of the CDR framework or region of both the VH and VL domain compared to a domain VH or VL derived from camelid, examples of which include, but are not limited to, camelid VH domains comprising the amino acid sequence shown as SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 77 or 88, and the camelid VL domains comprising the amino acid sequence shown as SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, 78, 89 or 149-164.
In other embodiments, "chimeric" antibody molecules comprising camelid-derived VH and VL domains (or engineered variants thereof) and one or more constant domains of a non-camelid antibody, for example, constant domains encoded by human ( or engineered variants thereof). In such embodiments it is preferred that both the VH domain and the VL domain are obtained from the same species of camelid, for example, both the VH and VL can be Lama glama or both VH and VL can be Lama pacos (before introduction of the variation of the engineered amino acid sequence). In such embodiments, both the VH and VL domains can be derived from a single animal, particularly a single animal that has been actively immunized with a human c-Met antigen.
As an alternative to engineered changes in the primary amino acid sequence of the VH and / or VL Camelidae domains, hypervariable loops derived from alternative camelid frameworks (ie, non-Camelidae}, for example, a human VH / VL framework, by grafting the CDR In particular, non-limiting, camelid-derived CDRs can be selected from among CDRs having the amino acid sequence shown as SEQ ID NOs: 1-21, 71-73 or 83-85 (heavy chain CDRs) or CDRs having the amino acid sequence shown as SEQ ID NOs: 22-42, 74-76, 86, 87 or 137-148 (light chain CDRs).
C-Met antibodies comprising camelid-derived VH and VL domains, or CDRs thereof, may have several different embodiments in which both a VH domain and a VL domain are present. The term "antibody" is used here in the broadest sense and includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (for example, bi-specific antibodies), as they exhibit the appropriate immunological specificity for a human cMet protein. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible minor mutations occurring. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include antibodies directed against different determinants (epitopes) on the antigen, each monoclonal antibody is directed against a single determinant or epitope on the antigen.
"Antibody fragments" comprise a portion of a full length antibody, generally binding to an antigen or variable domain thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, bi-specific Fab's, and Fv fragments, diabody, linear antibodies, single chain antibody molecules, a variable single chain fragment (scFv), antibodies of the multi-specific domain and antibodies formed from antibody fragments (see Holliger and Hudson, Nature Biotechnol. 23: 1126-36 (2005), the contents of which are incorporated herein by reference).
In non-limiting embodiments, cMet antibodies comprising camelid-derived VH and VL domains, or CDRs thereof, may comprise CHI domains and / or CL domains, the amino acid sequence of which is human At-amp or snhstanc.i. . Where the antigen-binding polypeptide of the invention is an antibody designed for human therapeutic use, it is typical for the entire antibody constant region, or at least part of it, to have a completely or substantially human amino acid sequence. For this reason, one or more or any combination of the CHI domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) can be completely or substantially human with respect to its amino acid sequence.
Advantageously, the CHI domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) can all have completely or substantially a human amino acid sequence. In the context of the constant region of a humanized chimeric antibody, or an antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 95%, or at least 97%, or at least 99% with a human constant region. The term "human amino acid sequence" in this context refers to an amino acid sequence that is encoded by a human immunoglobulin gene, which includes genes of germline lineage, rearranged and modified by snmatinamante mutation. The invention also contains new insides comprising domains contained in the "human" sequence that has been altered, by one or more additions, deletions or substitutions of amino acids with respect to the human sequence, except for those embodiments where the presence of a region of "completely human" hinge is expressly required.
The presence of a "completely human" hinge region in the c-Met antibodies present in the product combination or composition or in the multi-specific antibody can be beneficial both to minimize immunogenicity and to optimize the stability of the antibody.
As discussed elsewhere here, it is contemplated that one or more amino acid substitutions, insertions or deletions can be made within the constant region of the heavy and / or light chain, particularly within the Fc region. Amino acid substitutions can result in the substitution of the substituted amino acid with a different naturally occurring substituted amino acid, or with an unnatural or modified amino acid. Other structural modifications are also permitted, such as, for example, changes in the glycosylation pattern (for example, by the addition or deletion of N or O-linked glycosylation sites). Depending on the desired use of the antibody, it may be Ho aa -1 gold 1 moH -i par r ~> antI om-noi from i nvonr-ãn rpm ra 1 arãn a α its binding properties with respect to Fc receptors, for example, to modulate the effector function. For example, cysteine residue (s) can be introduced into the Fc region, thus allowing the formation of the interchain disulfide bond in this region. The homodimeric antibody generated in this way may have improved internalization capacity and / or complement-mediated cell death and increased antibody-dependent cell cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Alternatively, a c-Met antibody can be worked which has double Fc regions and can thus have complement analysis and increased ADCC capabilities. See Stevenson et al., AntiCancer Drug Design 3: 219-230 (1989). The invention also contemplates immuno-conjugates comprising an antibody as described herein conjugated to a cytotoxicity agent such as a chemotherapeutic agent, toxin (for example, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radio-conjugate). The Fc regions can also be engineered for half-life extension, as described by Chan and Carter, Nature Reviews: Immunology, Vol.10, pp301-316, 2010, incorporated herein by reference. Int-1 rnrnní r-Mpt vari ant-ps pm mip a r-pcriíín Pr * Á modified by engineered protein, as described here, may also exhibit an improvement in efficacy (for example, in the treatment of cancer), compared to a equivalent antibody (i.e., equivalent antigen binding properties) without modifying the Fc.
In yet another embodiment, the Fc region is modified to increase the antibody's ability to mediate antibody-dependent cell cytotoxicity (ADCC) and / or to increase the antibody's affinity for an Fcy receptor by modifying one or more amino acids.
In yet another embodiment, the glycosylation of an antibody antibody is modified. For example, an agglomerated antibody can be made (that is, the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the target c-Met antigen. Such carbohydrate modifications can be carried out by; for example, alteration of one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more glycosylation sites from the variable region framework to thereby eliminate glycosylation at that site. Such aglycosylation can increase
Also considered are variant c-Met antibodies having an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or a non-fucosylated antibody (as described by Natsume et al., Drug Design Development and Therapy, Vol.3 , pp7-16, 2009) or an antibody having enlarged bi-sectioned GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC activity of antibodies, typically producing a 10-fold increase in ADCC relative to an equivalent antibody comprising a "native" human Fc domain. Such carbohydrate modifications can be carried out, for example, by expressing the antibody in a host cell with altered glycosylation enzyme machinery (as described by Yamane-Ohnuki and Satoh, rnAbs 1: 3, 230-236, 2009).
Still other embodiments of c-Met antibodies may be lack of effector function, or because the Fc portion of the antibody is of an isotype that is naturally lacking effector function, or that exhibits significantly less potent effector function than IgGl, for example example, human IgG2 or human IgG4, or because the antibody's Fc function was engineered to reduce or substantially <=> 1 imi na FunrSn nmmn Hpcrribn <=> m Zlr-rnrrnv R T. et al., Eur. J. Immunol ., 1999, 29: 2613-2624.
In still other embodiments the Fc portion of the c-Met antibody can be engineered to facilitate preferential formation of bispecific antibodies, wherein two heavy chains of the antibody comprise pairs of different domains to form the Fc portion of the bispecific antibody. Examples of such modifications include the "knobs-into-hole" modifications described by Ridgway JB, Presta LG, Carter P., 'Knobs-into-holes'engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 Jul; 9 (7): 617-21 and Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, Carter P. An efficient route to human bispecific IgG. Nat Biotechnol. 1998 Jul; 16 (7): 677-81.
The invention may, in certain embodiments, contain chimeric Camelidae / human antibodies, and in particular chimeric antibodies in which the VH and VL domains are from complete camelid sequences (for example, Llama or alpaca) and the rest of the antibody is completely human sequence. C-Met antibodies can include antibodies comprising "humanized" or "germline" variants of VH and VL domains, or CDRs thereof, and chimeric camelid / human antibodies, where the VH and VL domains contain one or more amino acid substitutions in the framework regions compared to VH and VL domains obtained by active immunization of a camelid with a human c-Met antigen. Such "humanization" increases the% of the sequence identity with the human germline VH or VL domains by replacing improperly combined amino acid residues in a starting VH or VL Camelidae domain with the equivalent residue found in a VH or VL domain encoded by the human germ line.
C-Met antibodies can also be antibodies grafted onto CDR, where CDRs (or hypervariable loops) derived from a camelid antibody, for example, a camelid c-Met antibody increased by active immunization with human c-Met protein, or otherwise encoded by a camelid gene, are grafted onto a human VH and VL framework, with the remainder of the antibody also being entirely of human origin. Such c-Met antibodies grafted onto the CDR may contain CDRs having the amino acid sequence shown as SEQ ID NOs: 1-21, 71-73 or 83-85 (heavy chain CDRs) or CDRs having the amino acid sequence shown as SEQ ID NOs: 22-42, 74-76, 86, 87 or 137-148 (light chain CDRs).
Humanized, chimeric and onvorh c-Met antibodies in rHD rrvrri i br> car * i ma nart i rnl q rmpnt "P antibodies, comprising hypervariable bonds or CDRs derived from active immunization of camelids with a human c-Met antigen, can be rapidly produced using conventional recombinant DNA manipulation and expression techniques, making use of engineered prokaryotic and eukaryotic host cells to produce the polypeptide of interest and including, but not limited to, bacterial cells, yeast cells, mammalian cells, insect cells, and plant cells, some of which as described here and illustrated in the accompanying drawings.
Camelid-derived c-Met antibodies include variants in which the hypervariable loop (s) or CDR (s) of the VH domain and / or the VL domain are obtained from a conventional camelid antibody raised against the human c-Met, but in which at least one of said hypervariable loops or CDRs (derived from camelid) have been engineered to include one or more amino acid substitutions, additions or deletions, relative to the camelid-encoded sequence. Such changes include "humanization" of hypervariable ties / CDRs. Camelid-derived HVs / CDRs that have been engineered in this way can still exhibit an amino acid sequence that a camelid-encoded HV / CDR. In this context, the "substantial identity" may allow for no more than one, or no more than two amino acid sequences mismatched with the camelid-encoded HV / CDR. Particular embodiments of the c-Met antibody may contain humanized variants of the CDR sequences shown as SEQ ID NOs: 1-21, 71-73 or 83-85 (heavy chain CDRs) and / or humanized variants of the sequences shown as SEQ ID NOs: 22-42, 74-76, 86, 87 or 137-148 (light chain CDRs).
The camelid-derived c-Met antibodies provided herein can be of any isotype. Antibodies designed for human therapeutic use will typically be of the IgA, IgD, IgE IgG, IgM type, often of the IgG type, in which case they may belong to any of the four subclasses IgGl, IgG2a and b, IgG3 or IgG4. Within each of these subclasses it is permitted to make one or more substitutions, insertions or deletions of amino acid within the Fc portion, or to make structural modifications, for example, to increase or reduce the Fc-dependent functionalities.
Humanization (germinalization) of camelid-derived VH and VL domains
Conventional camelid antibodies provide an advantageous starting point for the preparation of antibodies useful as human therapeutic agents due to the following factors, discussed in US 12/497, which is incorporated here as a reference: 1) High sequence homology between VH and VL domains of camelids and their human counterparts; 2) High degree of structural homology between the CDRs of the camelid VH and VL domains and their human counterparts (ie human canonical fold structures and human type combinations of canonical folds).
The camelid platform (for example, llama) also provides a significant advantage in terms of the functional diversity of the c-Met antibodies that can be obtained.
The utility of c-Met antibodies comprising camelid VH and / or camelid VL domains can be further enhanced by "humanizing" or "germinalizing" the natural camelid VH and VL domains, for example, to make them less immunogenic in a human host . The total objective of humanization is to produce a molecule in which the VH and VL domains exhibit minimal immunogenicity i nr, i irí Hnn hnmann Anmiantri rptpnHri the specificity and affinity of the antigen binding site formed by the VH and VL domains.
An approach to humanization, called "germinalization", involves engineering the amino acid sequence of a camelid VH or VL domain to bring it closer to the sequence of a human VH or VL domain.
The determination of homology between a VH (or VL) domain and human VH (or VL) domains is a critical step in the humanization process, both for the selection of camelid amino acid residues to be changed (in a given VH or VL domain) ) and for the selection of substitution of the appropriate amino acid residue (s).
An approach to the humanization of conventional camelid antibodies has been developed based on the alignment of a large number of new sequences of the camelid VH (and VL) domain, typically mutation-changed VH (or VL) domains that are known to link a target antigen, VH (or VL) sequences with human germline, consensus sequences with human VH (and VL), as well as germline sequence information for pacos llama.
The following passages describe the principles that "camelid" amino acids for substitution in a camelid-derived VH or VL domain or a CDR thereof, and (ii) select the replacement of "human" amino acid residues to replace in, when humanizing any camelid VH (or VL) domain. This approach can be used to prepare humanized variants of camelid-derived CDRs having the amino acid sequence shown as SEQ ID NOs: 1-21, 71-73 or 83-85 (heavy chain CDRs) or having the amino acid sequence shown as SEQ ID NOs: 22-42, 74-76, 86, 87 OR 137-148 (light chain CDRs), and also for the humanization of camelid-derived VH domains having the sequences shown as SEQ ID NOs: 45-51, 77 or 88 camelid-derived VL domains having the sequences shown as SEQ ID NOs: 52-58, 78, 89 OR 149-164.
Step 1. Select family and human member (of germ line) of this family that shows higher homology / identity with the mature camelid sequence to be humanized. A general procedure for identifying the closest match to the human germline for any given camelid VH (or VL) domain is described below.
Step 2. Select the family member -vm n no 1 dc Non i fi nn see the line rrí => m rrarm i na 1 nnnhra Preferably this is the germ line with the highest homology or another member of the germline family of the same family.
Step 3. Identify the preferred positions considered for germinalization based on the amino acid utilization table for the camelid germ line that is closest to the selected human germ line.
Step 4. Try to exchange amino acids in the camelid germ line that deviates from the nearest human germ line; germinalization of FR residues is preferred over CDR residues. The. Preferred are positions that are deviating from the selected human germline used for counter germline, for which the amino acid found in the camelid sequence does not match the selected germline and is not found in other germ lines of the same subclass (both for V as well as FR amino acids encoded by J). B. Positions that are deviating from the family member of the selected human germ line, but which are used in other germ lines of the same family can also be directed in the germinalization process. due to additional somatic mutations) for the selected human germline can also be targeted.
The following approach can be used to determine the closest match to the human germline for a given camelid VH (or VL) domain:
Before analyzing the sequence identity between VH and VL, Camelidae and human canonical folds can be determined first, which allows the identification of the family of segments of the human germ line with the identical combination of canonical folds for Hl and H2 or Ll and L2 (and L3). Subsequently, the member of the human germline family that has the highest degree of sequence homology with the variable Camelidae region of interest can be chosen for the sequence homology score. The determination of the Chothia canonical classes of the hypervariable loops Ll, L2, L3, Hl and H2 can be carried out with the computer tools available publicly on the website www.bioinf.org.uk/abs/chothia.html. The provision of program information shows the requirements of the key residue in a data file. In these data files, the positions of the key residue are shown with the amino acids allowed in each position. The sequence of the variable region of the antibody is given as an entry and is the ”1 i rxvirro-i nmo o om ior r * n a o n -i noTiri / Consensus to satisfy the Kabat numbering scheme. The analysis of canonical folds uses a set of models of the key residue derived by an automated method developed by Martin and Thornton (Martin et al., J. Mol. Biol. 263: 800-815 (1996)). The boundaries of the regions of the individual framework can be designated using the IMGT numbering scheme, which is an adaptation of Chothia's numbering scheme (Lefranc et al., NAR 27: 209-212 (1999); http: //imgt.cines .fr).
With segment V of the particular known human germline, which uses the same combination of canonical folds for Hl and H2 or Ll and L2 (and L3), the member of the family that best matches in terms of sequence homology can be determined. The percentage of sequence identity between the amino acid sequences of the VH and VL Camelidae domain framework and corresponding sequences encoded by the human germline can be determined using bioinformatics tools, but manual sequence alignment could also be used. Human immunoglobulin sequences can be identified from various protein databases, such as VBase (http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun / Honegger / hb bn database • / / hmm; k ^ 4 iirri 7 ) rh / anbi k »/ nrlw I Qom cs / slept 4 noa
To compare human sequences with the V regions of the VH or VL Camelidae domains a sequence alignment algorithm as available via websites like www.expasy.ch/tools/tfalign can be used, but manual alignment can also be performed with a limited set of strings. Sequences of light and heavy chain of the human germ line of families with the same combinations of canonical folds and with the highest degree of homology with the regions of framework 1, 2, and 3 of each chain can be selected and compared with the variable region Camelidae of interest; FR4 is also verified against the human germline regions JH and JK or JL.
Note that in the youngest percentage of the total homology of the sequence of the FR1, FR2 and FR3 residues, they are evaluated using the closest combination of sequence of the human germline family with the identical combination of canonical folds. Only residues other than the closest combination or other members of the same family with the same combination of canonical folds are classified (NB excluding any differences coded by the initiators). However, for the purpose of humanization, residues in the regions of the human germline framework, which does not have the same combination of canonical folds, can be considered for humanization, despite the fact that they are classified as "negative" according to the conditions described above . This assumption and based on the "mix and match" approach to humanization, in which each of FR1, FR2, FR3 and FR4 is compared separately with its closest matching human germline sequence and the humanized molecule, therefore contains hence a combination of different RFs as done by Qu and colleagues (Qu et al., Clin. Cancer Res. 5: 3095-3100 (1999)) and Ono and colleagues (Ono et al., Mol. Immunol. 36: 387 -395 (1999)).
Just as an example, it is contemplated that humanized variants of the VH domains having the amino acid sequence shown as SEQ ID Nos: 45-51, 77 or 88 may include variants in which the amino acid residue (s) occurring in a or more of the positions listed in the following table are / are replaced with an amino acid residue that occurs at the equivalent position in a human VH domain, for example, a VH domain encoded by the human germline. Appropriate amino acid substitutions can be derived following the general humanization protocol described above.
Table • List of risks and amino acids that can be substituted during the germinalization (humanization) of the listed VH domains. For each so-called VH domain, the listed amino acid residues are numbered according to the Kabat numbering system.
* note that the replacement of residues 54 and 55 is for the purpose of removing a deamidation site, not for human germinalization as such.
Just as an example, it is contemplated that humanized variants of the VL domains having the amino acid sequence shown as SEQ ID Nos: 52-58, 78, 89 or 137-148 may include variants in which the residue (s) of amino acid occurring at one or more of the positions listed in the following table is / are replaced with an amino acid residue occurring at the equivalent position in a human VL domain, for example, a VL domain encoded by the human germline. Appropriate amino acid substitutions can be derived by the following general humanization protocol described above.
Table 4: List of amino acid residue positions that can be replaced during germinalization (humanization) of the listed VL domains. For each so-called VL domain, the listed amino acid residues are numbered according to the Kabat numbering system.

Cross Antibodies for Competition
Monoclonal antibodies or antigen-binding fragments thereof that "cross-compete" with the molecules described here are those that bind to human c-Met at the site (s) that are identical to, or overlap, with the site (s) where the cMet antibodies present bind. Competitive monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition test. For example, a sample of purified or partially purified human c-Met can be attached to a solid support. Then, an antibody compound or antigen-binding fragment of the same invention and a monoclonal antibody or antigen-binding fragment of the same suspected to be able to compete with such an antibody compound are added. One of the two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate and discrete sites in cMet, the labeled compound will bind at the same level whether or not the suspect competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be decreased. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind. For the purposes of the present invention, competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the compounds of the present antibody to c-Met by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Details of the procedures for performing such competition tests are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pages 567 -569, ISBN 0-87969-314-2. Such tests can be made quantitative using purified antibodies. A standard curve is established by the antibody titration used both as a label and as a competitor. The ability of an unlabeled competing monoclonal antibody or antigen binding fragment thereof to inhibit binding of the labeled molecule to the plate is titrated. The results are plotted, and the concentrations required to achieve the desired degree of binding inhibition are compared. Polynucleotides encoding c-Met antibodies
The invention also provides polynucleotide molecules encoding the c-Met antibodies present in a combination or composition of the product (or at least the antigen-binding portions thereof) or encoding the antigen-binding regions of the multi-specific antibody, also vectors cells containing nucleotide sequences encoding the c-Met antibodies of the invention operably linked to regulatory sequences that allow expression of the antigen-binding polypeptide in a host cell or free cell expression system, and a host cell or expression system of free cell containing this expression vector.
In particular embodiments, the polynucleotide of the c-Met antibodies present in a combination or composition of the product (or at least those antigen-binding the multi-specific antibody may comprise one or more of the nucleotide sequences shown as SEQ ID NOs: 59-70, 79-82, 90, 91, 122-135 or 165-180, sequences which encode VH or VL domains of c-Met antibodies, or a variant sequence that encodes a functional VH or VL domain of a cMet antibody , wherein said variant sequence exhibits at least 80%, 85%, 90%, 95%, 97% or 99% of the sequence identity when ideally aligned with one of SEQ ID NOs: 59-70, 79-82, 90, 91, 122-135 or 165-180 In this context,% of the sequence identity between two polynucleotide sequences can be determined by comparing these two sequences ideally aligned and in which the polynucleotide sequence to be compared can comprise additions or deletions with respect to the reference sequence for a optimal alignment between the two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide residue is identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window by multiplying the result obtained by 100 in order to obtain the percentage of identity between these two strings. For example, it is (Tatusova et al, "Blast 2 sequences a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250) available at http://www.ncbi.nlm.nih.gov / gorf / bl2.html, the parameters used being those given by the faults (in particular for the parameters "open gap penalty": 5, and "extension gap penalty": 2; the matrix chosen being, for example, the matrix "BLOSUM 62 "proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.
Polynucleotide molecules encoding the c-Met antibodies present in the product combination or composition (or at least the antigen-binding portions thereof) or encoding the antigen-binding regions of the multi-specific antibody include, for example, DNA molecules recombinant. The terms "nucleic acid", "polynucleotide" or a "polynucleotide molecule" are used interchangeably here and refer to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule can be described here according to the 3 '. In some embodiments of the invention, nucleic acids or polynucleotides are isolated. "This term, when applied to a nucleic acid molecule, refers to a nucleic acid molecule that is separated from the sequences with which it is immediately contiguous in the genome. naturally occurring in the organism it originated in. For example, an "isolated nucleic acid" can comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into a cell's genomic DNA prokaryotic or eukaryotic or a non-human host organism When applied to RNA, the term "isolated polynucleotide" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to a molecule of RNA that has been purified / separated from the other nucleic acids with which it would be associated in its natural state (that is, in cells or tissue). isolated inucleotide (or DNA or RNA) can also represent a molecule produced directly by biological or synthetic means and separated from the other components present during its production.
For recombinant production of a c-Met antibody encoding it, it can be prepared (using standard molecular biology techniques) and inserted into a replicable vector for expression in a chosen cell, or a cell-free expression system. Suitable host cells can be prokaryotic, yeast, or higher eukaryotic cells, specifically mammalian cells. Examples of usable mammalian host cell lines are, monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lineage (293 or 293 cells subcloned in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse sertose cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL 8287) or NS0 (HPA culture collections no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); rat buffalo liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); TTD liver cells o n c c m - »m o n a rm iri« A r n rrr (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma strain (Hep G2), as well as a DSM's PERC-6 cell strain. Expression vectors suitable for use in each of these host cells are also generally known in the art.
It should be noted that the term "host cell" generally refers to a cutivated cell line. All humans within which an expression vector encoding an antigen-binding polypeptide according to the invention has been introduced are explicitly excluded from the definition of a "host cell". Antibody production
A method of producing a c-Met antibody of the invention can comprise culturing a host cell (or cell-free expression system) containing polynuceotide (for example, an expression vector) encoding the c-Met antibody under conditions that allow for expression of the c-Met antibody, and recovering the expressed cMet antibody. This recombinant expression process can be used for large-scale production of c-Met antibodies according to the invention, including monoclonal antibodies designed for therapeutic use and production suitable for large-scale manufacture of recombinant antibodies suitable for therapeutic use in vivo are generally available in the art and are well known to those skilled in the art. Therapeutic utility of cMet antibody combinations
The product combinations or compositions, or the multi-specific c-Met antibodies provided herein can be used in the treatment of both HGF-dependent and HGF-independent cancers.
Improper activation of c-Met can be induced by specific genetic lesions, positive transcriptional regulation or ligand-dependent autocrine or parocrine-dependent mechanisms (Comoglio et al, Nature Reviews Drug Discovery, 7: 504-516,2008). HGF-dependent and HGF-independent cancers that can be treated with product combinations or compositions, or multi-specific c-Met antibodies include, but are not limited to gastric carcinomas, esophageal carcinomas, medulloblastomas, liver cancer metastases colon, renal papillary carcinoma, squamous cell carcinomas of the head and neck, carcinomas of the thyroid, ovary, pancreas, protrate, renal cell, hepatocellular, osteosarcomas.
The term "treat" or "treatment" means to interrupt, suspend, control, stop, slowly reduce the severity of a symptom, disorder, condition or disease, but it does not necessarily involve the total elimination of all related symptoms, conditions or disorders with the disease.
For human therapeutic use, the product combinations or compositions, or the multispecific c-Met antibodies described herein can be administered to a human subject in need of treatment in an "effective amount". The term "effective amount" refers to the amount or dose of a c-Met antibody that, using the administration of a single or multiple dose to a human patient, provides therapeutic efficacy in the treatment of the disease. Therapeutically effective amounts of the c-Met antibody product combinations or compositions, or the multi-specific c-Met antibodies can comprise an amount in the range of about 0.1 mg / kg to about 20 mg / kg per single dose. An effective therapeutic amount for any individual patient can be determined by the medical professional by monitoring the effect of the c-Met antibody on a biomarker, such as c-Met on the surface of the tumor cell, etc. The amount of antibody administered at any given point in time can be varied so that ideal amounts of the c-Met antibody, if used alone or in combination with any other therapeutic agent, are administered during the course of treatment.
It is also contemplated how to administer the combinations or compositions of the product, or the multispecific c-Met antibodies described herein, or pharmaceutical compositions comprising such antibodies, in combination with any other cancer treatment, as a combination therapy. Pharmaceutical compositions
The scope of the invention includes pharmaceutical compositions, containing a combination of the c-Met antibodies of the invention, or antigen fragments thereof, formulated with one or more acceptable vehicles or excipients. Such compositions can include any of the combinations of the cMet antibodies described herein. For example, a pharmaceutical composition of the invention of the invention may comprise a combination of antibodies that bind to different epitopes in human c-Met, for example, an antibody binding to the SEMA domain of human c-Met combined with an antibody that binds within the PSI-IPT domain of the human c-Met. comprising a combination or mixture of a first antibody, or antigen-binding fragment thereof which is a 48A2 variant, or 4 8A2 as defined herein, or an antibody that competes with the reference antibody 48A2, or an antibody that binds to the same epitope in human c-Met as the reference antibody 48A2 and a second antibody, or antigen-binding fragment thereof that is a 36C4, or 36C4 variant as defined here, or an antibody that competes with the 36C4 reference antibody, or an antibody that binds to the same epitope on human c-Met as the reference antibody 36C4. The pharmaceutical composition can comprise a first antibody, or antigen-binding fragment thereof that is 48A2, or a variant of 48A2 as defined herein, or an antibody that competes with the reference antibody 48A2, or antibody that binds to it epitope on human c-Met as the reference antibody 48A2 and a second antibody, or antigen-binding fragment that is 36C4, or a variant of 36C4 as defined herein, or an antibody that competes with the reference antibody 36C4, or a antibody that binds to the same epitope in human c-Met as the reference antibody 36C4 mixed with one or more acceptable vehicles or excipients. comprise a first antibody, or antigen-binding fragment thereof that is 4 8A2, or a variant of 4 8A2 as defined herein, or an antibody that competes with the reference antibody, or an antibody that binds to the same epitope in c -Human met as the reference antibody 48A2 and a second antibody, or antigen binding fragment thereof which is 36C4, or a variant of 3 6C4 as defined herein, or an antibody that competes with the reference antibody 36C4, or a antibody that binds to the same epitope in human c-Met as the reference antibody 36C4, wherein the first and second antibodies are packed separately, rather than mixed.
Techniques for formulating monoclonal antibodies for human therapeutic use are well known in the art and are reviewed in, for example, Wang et al. , Journal of Pharmaceutical Sciences, Vol.96, ppl-26, 2007. Brief description of the drawings
The invention will also be understood with reference to the following experimental examples and the accompanying Figures in which:
Figure 1. MKN-45 specific immune response in pre-immune (day 0) and post-immune (day 45) sera from llama cells immunized with MKN-45, as measured by f 1 iivn cytometry
Figure 2. The immune response to recombinant c-Met in pre-immune (day 0) and post-immune (day 45) sera from llamas immunized with MKN-45 cells, as measured by ELISA.
Figure 3. Competition test showing periplasmic extracts containing Fab competing with terminally biotinylated HGF (25 ng / ml) for binding to c-Met captured via the C-terminal Fc portion.
Figure 4. ELISA illustrating binding of antibody 40B8 to the IPT1-2 domain of c-Met (A) and binding of 36C4 to the SEMA domain of c-Met (B).
Figure 5. The results of a spread test using HPAF cells demonstrating HGF-induced spread inhibition by the 38H10 antibody in a dose-dependent mode (top panel). No agonistic effects were observed compared to the control only medium.
Figure 6. An ELISA-based competition test illustrating the degree of competition between antibodies and HGF for binding of c-Met at different antibody concentrations. The percentage competition was calculated compared to control antibodies.
Figure 7. Proliferation test using BxPC3 cells. Chimeric 224G11 is C224G11. (A) Antibody-induced proliferation as a percentage of maximum effect at 75 ng / ml
HP MΠFÍRi The pfp> -i t of the HGF-induced ATTI nrol i fp.rrann ant as compared to the maximum effect at 75 ng / ml HGF.
Figure 8. Agonism as measured in a phosphorylation test using NSCLC A549 cells. The percentage of antibody-induced phosphorylation of c-Met is expressed as a percentage of phosphorylation induced by 100ng / ml HGF. Murine 224G11 (m224G11) and chimeric 224G11 (c224Gll) were included as positive controls and U16 antibody was included as a negative control.
Figure 9. Antagonism as measured in a phosphorylation test using A549 cells. Inhibition of HGF-induced phosphorylation of c-Met by antibodies is indicated as a percentage compared to the maximum effect of 100 ng / ml HGF alone in A549 cells. Chimeric 224G11 (c224G11) was included as a positive control and U16 antibody as a negative control.
Figure 10. Blocking independent HGF activation measured in a phosphorylation test using MKN-45 cells. The inhibition of autophosphorylation in MKN-45 cells by antibodies was compared with the negative control U16.1, where the inhibition by U16.1 was fixed as 0%.
Figure 11. Antibody-induced ADCC in MKN45 cells using Dead-Cell Protease Kit (cytotoxicity test xi_ _J m—. __ z "i“ I - TM -n TJ - -. -. X.. - 1 £. ~ _ __ _ _ - _ J ~ - - specific TJ compared to the negative isotype control.
Figure 12. 36C4 Potelligent ™ induced ADCC in NCI-H441 cells expressed as the percentage of cell lysis as measured using a 51Cr release test.
Figure 13. In vivo effect of 36C4 enhanced by ADCC on MKN-45 xenografts with twice a week injections of mAb.
Figure 14A-B. Surface plasmon resonance of 36C4 and 48A2 for binding to non-overlapping epitopes. Bonding is observed for the Met: 48A2 complex only (A) and for the Met: 36C4 complex only (B).
Figure 15. Alignment of human c-Met and Llama glama amino acid sequences.
Figure 16A-B. Domain mapping of mAbs using chimeric c-Met ECD. 36C4 binding to human c-Met (WT) and the binding indicating human IPT1-4 / llama for the SEMAPSI region (A). Binding of 13E6 mAb to human c-Met and to llama / human IPT1-4 (B).
Figure 17. Inhibition of autophosphorylation using combinations of c-Met mAbs in MKN-45 cells.
Figure 18. The results of a phosphorylation test using combinations of c-Met mAbs in NSCLC A549 cells showing agonistic effects (A) and positive antagonistic effects.
Figure 19. U87 MG xenograft experiment in vivo testing the effects of administration of 30 mg / kg 36C4 on tumor growth versus the effect of administration of 30 mg / kg of C224G11.
Figure 20. Phosphorylation test using germinalized 36C4 mAbs in A549 cells showing agonism (A) and antagonism (B). U16 is the isotype control and C224G11 is the positive control.
Figure 21. Stability of PBS of 36C4 germline variants at various temperatures. Functionality tests were performed using surface plasmon resonance in germinalized 6C4 mAbs after incubation in PBS at 4 ° C, RT and 37 ° C for up to 56 days.
Figure 22. Thermotolerance of 36C4 (A) and 48A2 (B) of germ line. Functionality investigated using surface plasmon resonance after incubation at different temperatures for 1 h.
Figure 23. Schematic illustration of the structure of chimeric c-Met llamahuman constructs prepared for: (A) mapping of mAb binding peptide (for example, indicates human c-Met (hS) sequence. The relative positions of the signal sequence, domain SEMA, PSI domain and IPT domains 1, 2, 3 and 4 are indicated; (B) mapping of mAb binding peptide (eg 48A2) to the PSI-IPT1 domain of c-Met. Light gray shading indicates c sequence -Meta llama, dark gray shading indicates human c-Met sequence, relative positions of signal sequences, SEMA domain, PSI domain and IPT domains 1, 2, 3 and 4 are indicated.
Figure 24. A test for negatively regulating total c-Met protein on the surface of MKN-45 cells following treatment with several c-Met mAbs in concentrations of 1 pg / ml or 10 pg / ml. The results are expressed as a total percentage of negative c-Met regulation.
Figure 25. The amino acid sequence of the extracellular portion of human c-Met, illustrating the positions of the SEMA domain and IPT domains.
Figure 26. Agonistic properties of different combinations of mAbs in HSC-dependent NSCLC A549 cells in a phosphorylation test. (A) Agonism by two mAbs binding to non-overlapping epitopes on the SEMA domain of human c-Met. (B) Agonism by two mAbs linking two
Two mAbs agonism linking non-overlapping epitopes over the IPT domain. UI 6 is the IgGl and C224G11 isotype control the mAb reference. 100 ng / ml HGF were used as the maximum effect (100%) and used to compare with the effect of mAbs.
Figure 27. Antagonistic effects of mAb combinations in MKN-45 autophosphorylated cells in a phosphorylation test. (A) Two SEMA ligands, linking non-overlapping epitopes, blocking auto phosphorylation, as compared to 36C4 and 48A2. (B) Comparison of the combination of a SEMA linker and an IPT linker versus the combination of 36C4 and 48A2. (C) Comparison of two IPT ligands, recognizing non-overlapping epitopes, versus the combination of 36C4 and 48A2. U16 is the IgGl isotype control used as the 0% reference and C224G11 the mAb reference.
Figure 28. Inhibition of HPAF cell spreading in the presence of 40 ng / ml HGF.
Figure 29. Illustrates the configuration of an exemplary ELISA to demonstrate bi-specificity. The exemplary bispecific antibody comprises a VH / VÀ binding site (for example, derived from a 36C4 or 20F1 antibody) that specifically recognizes the SEMA domain of cMet and a VH / VK binding site (for example, antibody derived 38H10
MET. In the test, SEMA is coated on the ELISA plate and the specific Ab bi is specifically detected with an anti-human CK antibody.
Figure 30. Illustrates SEMA binding of mAb mixtures detected with anti-human Fc antibody. Cultures of HEK cells transfected with plasmid mixtures encoding HC and LC of 36C4 / 20F1 and 38H10 / 40B8 were purified with protein A and tested in two concentrations. Parental mAbs 40B8 and 38H10, both specific for IPT, and 36C4 and 20F1, specific for SEMA, were included below for the isotype control (U16.1).
Figure 31. Illustrates SEMA binding of bi-specific mAbs as detected with anti-CK antibody. Cultures of HEK cells transfected with plasmid mixtures encoding HC and LC of 36C4 / 20F1 and 38H10 / 40B8 were purified with protein A and tested in two concentrations. Parental mAbs 40B8 and 38H10, both specific for IPT, and 36C4 and 20F1, specific for SEMA, were included close to the isotype control (U16.1).
Figure 32. Illustrates a CBB-colored PAGE of purified forced wrong combinations of protein A (encoded A) and Kappa-Select (encoded K) or LambdaSelect (encoded L) or both (encoded LK) (1 4) or bispecific (5 and 6). CBB gels are shown from reduced samples (panel A) or unreduced samples (panel B). Sample 1 is VH36C4 + VK40B8, sample 2 VH40B8 + VL36C4, sample 3 VH36C4 + VK38H10, sample 4 VH38H10 + VL36C4, sample 5 VHVL36C4 + VHVK40B8 bi specific and sample 6 VHVL36C4 + VHVK38H10 bi.
Figure 33. Illustrates SEMA binding of all purified combinations, as detected with (A) anti-CK and (B) anti-Fc antibodies. The forced wrong combinations of VH and VL (transfection 1 to 4) giving paired mAbs were not functional in recognizing the immobilized SEMA domain. The bi-specific purified samples of 38H10 and 40B8 gave high signs of binding when detected with anti-CK and anti-Fc antibodies.
Figure 34. Illustrates SEMA binding of samples taken during purifications as detected with (A) anti-CK and (B) anti-Fc antibodies. Enrichment during purification can be observed in ELISA with the detection of anti-kappa antibody (A), confirming that each step signals after purification in kappa beads as compared to lambda beads, suggesting that the parental antibodies have been removed.
Figure 35. Illustrates (A) theoretical combinations of heavy and light chain pairs produced by hybrid hybridomas and (B) combinations obtained by subsequent purification of Kappa-Select and Lambda-Select. The two parental antibodies are shown in blue and yellow while the bi-specific antibody with non-promiscuous VL domains is surrounded. Incorporation as a reference
Several publications are cited in the foregoing description and from beginning to end of the following examples, each of which is incorporated herein by reference in its entirety. Examples
The invention will be further understood with reference to the following non-limiting experimental examples. Example 1: Immunization of llamas
Immunization of llamas and collection of peripheral blood lymphocytes (PBLs), as well as subsequent RNA extraction and amplification of antibody fragments, was performed as described by De Haard and colleagues (De Haard H, et al., JBC. 274 : 18218-30, 1999). Eight llamas were MKN-45 human gastric (DMSZ, ACC409) (c-Met overexpression was confirmed by flow cytometry using PE-conjugated anti-HGFR antibody (R&D systems, cat in FAB3582P)). Two others were immunized with NCIH441 cells from the lung cell line. Llamas were immunized with intramuscular injections in the neck once a week for a period of six weeks. Approximately 107 cells were injected into the neck muscles and Freund's incomplete adjuvant was injected into a second region located a few centimeters from the cell injection site.
10 ml blood samples were collected before and after immunization to investigate the immune response. Three or four days after the last immunization, 400 ml of blood was collected and RNA extracted from PBLs prepared using a Ficoll-Paque gradient and the method described by Chomczynski P et al. (Anal. Biochem. 162: 156-159, 1987). The average RNA yield was 450 pg. The extracted RNA was then used for random cDNA synthesis and PCR amplification of the V regions of the heavy and light chains (VÀ and VK) in order to construct the Fab-containing phagemid libraries as described by De Haard H, et al. (Biol. Chem. 274, 1999). The resulting libraries show good levels of 2 -i-JΠ ..- IA8X
The immune response to MKN-45 cells or NCI-H441 cells was investigated using flow cytometry. 100 µl / well of diluted sera was added onto the cells (2x105 cells / well) and incubated for 30 minutes at 4 ° C. After washing with PBS and 1% BSA, 0.1 pg / 100pl / well of FITC-conjugated goat anti-llama antibody (BETHYL, # A160-100F) was added and incubated for 30 minutes at 4 ° C. After washing with PBS and 1% BSA the results were read on a Calibur FACS and the fluorescent medium was plotted against the dilutions of the sera (Figure 1).
The specific immune response to c-Met was determined using an ELISA with immobilized recombinant c-Met (R&D systems, 358-MT / CF) using pre- and post-immune sera (Day 0 and Day 45 respectively). IgGl of llama bound to immobilized c-Met was detected using an anti-mouse mud IgGl (Daley LP, et al. Clin. Diagn. Lab Immunol. 12: 380-386, 2005) an anti-monkey mouse antibody conjugated to HRP (Jackson). Figure 2 shows the immune response of 4 out of 10 immunized llamas. A similar immune response was observed for the other 4 immunized with MKN-45 cells, but not for llamas immunized with NCI-H441 cells. c-Met
Fabs expressing phage were produced according to standard protocols and still selected in immobilized recombinant dimeric c-Met (R&D systems, 358-MT / CF) or recombinant extracellular domain of c-Met. Total elution of the c-Met binding phage with trypsin was performed according to standard phage display protocols.
Two or three series of selection were performed to enrich the specific c-Met Fabs expressed by the phage. Individual colonies were isolated and periplasmic fractions (peris) were produced by inducing IPTG from all libraries according to standard protocols.
Screening of c-Met-specific Fabs for competition with mature HGF for binding to immobilized c-Met was performed using an ELISA-based competition test. 2 pg / ml of goat anti-human Fcy antibody (Jackson) were immobilized on a maxisorb plate and, after blocking with 1% casein in PBS for 2 h, 100 ng / ml of recombinant dimeric c-Met was added and incubated for 1 h at room temperature. After washing, 50 µl of the Fab containing profiles was added and allowed to bind to the captured c-Met, before 25 ng / ml of mature N-terminally biotinylated HGF (R&D systems. 294-HGN / CF) was added. Nterminal biotinylation was performed according to the protocol provided by Thermo Scientific with a 5-fold excess of NHS-LC biotin in a 50 mM phosphate buffer (pH 6.5) at 4 ° C for 24 h. The mature biotinylated HGF was incubated at room temperature for 1 h before washing and adding streptavidin conjugated to horseradish (strep-HRP) and incubated for an additional hour. TMB was added and the plate read at 620 nm. A non-relevant periplasmic extract and a 50-fold excess of cold (non-biotinylated) HGF was included as a positive control on all plates. An example of profiles containing Fab competing with HGF is given in Figure 3.
Clones competing with HGF were sequenced in the VH and VL regions and divided into VH families based on the CDR3 sequence in the VH. These VH families were further tested with Surface plasmon resonance (SPR) for dissociation (kOff) and recognition of SEMA-PSI or extracellular domain of c-Met (Decoy). Between 1000-2000 RU of dimeric c-Met, SEMA-PSI or c-Met Decoy were immobilized on a VIA chip with copulation of amine in sodium acetate buffer (pH 4.5). Profiles containing Fab were added at a flow rate of 30 µl / min. and the
Fabs were considered to be binding if an increased RU sample. Table 8 summarizes domain recognition and kOff for different VH families.
Several VH families recognized the SEMA-PSI domain, while others recognized only the c-Met Decoy. The Fabs 5 had a kOff in the range of 10'3 10'4 s'1, with the best (12G4) having a koff of 1.3 x 10'4 s'1.
The VH and VL domains of the antagonistic clones were fused with IgGl constant domains and with human CK domains and CÀ domains and produced as two-valent monoclonal antibodies in the system described in patent application WO 2009/145606 with expression yields of 1530 pg / ml after purification of protein A. Table 5: CDR sequences of antagonistic antibodies and variants of germline lineage (according to 15 Kabat numbering)




Table 6: Amino acid sequences of the heavy and light chain domains of the selected antagonistic Fabs and affinity variants




Table 7: Nucleotide sequences encoding variable heavy and light chain domains of the selected antagonistic Fabs.








Example 3: Mapping of the Ecto-domains epitope other than c-Met (Decoy, SEMA, SEMAPSI, SEMA-PSI-IPTl-2 and IPT3-4, (C. Basilico et al., J Biol. Chem. 283: 21267- 2127, 2008) were immobilized (1 pg / ml) in a maxisorb plate in PBS overnight at 4 ° C. The antibodies (mAbs) were added in three dilutions of 1 pg / ml and allowed to bind for 1 h at room temperature. The binding was developed with HRP-conjugated Protein A and TMB and read at 450 nm after completion of the reaction with H2SO4.Based on the binding results, the mAbs can be several mAbs that bound only to c-Met Decoy and not for any of the other domains tested (Table 8) Some antibodies binding only to c-Met Decoy can bind to the IPT 2-3 region or to a conformational epitope not shown in the recombinant c-Met protein fragments. binding of the 40B8 antibody to the IPT1-2 domain is shown in Figure 4A and 36C4 binding to the SEMA domain in Figure 4B. c-Met nium for antagonistic mAbs and corresponding Fab shutdown rates mAb Recognition of the koff domain (IO'4 s-1) 12G4 IPT1-2 1.3 13E6 Decoy 6.5 20F1 SEMA 69 20A11 Decoy 9 38H10 IPTl-2 12 36C4 SEMA 6.4 40B8 IPTl-2 13 34H7 SEMA 16 Example 4: Scattering test Human pancreatic cancer cells with serum deficiency (HPAF) were plated in 96 well plates, 7000 cells / well. On day 2, antibodies were added in triplicates at concentrations of 30, 10, 3 and 1 pg / ml, and incubated with the cells for 30 minutes before 1.25 ng / ml HGF / well was added. HPAF cells were also incubated with antibodies in the absence of HGF. On day 3, the cells were fixed and stained with violet crystal. The scattering quantity score was made three times and by two different people.
The results showed a dose-dependent inhibition of the scattering induced by HGF by mAbs, with strong blocking for eight antibodies from the 13 tested, of which five (12G4, 20A11, 38H10, 36C4 and 40B8) showed complete spread blocking with 30 pg / ml. All eight antagonistic mAbs (12G4, 13E6, 20F1, 20A11, 38H10, 34H7, 36C4 and 40B8) were also devoid of agonistic effects with 30 pg / ml in the absence of HGF. Figure 5 shows an example of the 38H10 spreading results in the presence and absence of HGF compared to the medium control and the HGF control. Example 5: Cross-reactivity for c-Met Rhesus and mouse
Cross-reactivity to ECD c-Met Rhesus (Maccaca mulatta, US20090191580_5) and mouse c-Met (systems r-a 1nr> • tí97fni pm a P.T.TÍIA <" <=>
ECD Rhesus was immobilized in PBS (1 pg / ml) in a 96 well maxisorb plate and incubated at 4 ° C overnight. After blocking with 1% casein in PBS, antibodies in dilutions starting at 10 µg / ml were added and allowed to bind for 1 h. at room temperature. The plate was washed and a goat anti-human Fey antibody (Jackson) was added and incubated for 1 h. at room temperature. After washing, TMB was added and the plate read at 620 nm.
Since the mouse c-Met also contained an Fc portion, the mAbs (2 pg / ml) were immobilized on a 96-well maxisorb plate overnight at 4 ° C and, after blocking, 100 ng / ml of c -Met of mice were added and incubated for 1 h. at room temperature. An HRP-conjugated mouse anti-His antibody (Serotech) was added and incubated for 1 h. at room temperature. After washing, TMB was added and the plate read at 620 nm. A biotinylated goat anti-mouse c-Met antibody revealed with strep-HRP was used as a positive control for mouse c-Met.
No significant (> 10-fold) link to mouse c-Met was observed for any of the mAbs.
All six mAbs tested showed reactivity vii 73Ha nara Rm r'-McatPhosnβ πnm an identical miacp linarãn compared to human c-Met ECD (Decoy) (Table 9). Table 9. EC50 (nM) of mAbs binding to Rhesus or human c-Met (Decoy) Human Rhesus mAb 38H10 0.17 0.19 40B8 0.13 0.14 36C4 0.14 0.13 2 0 All 3.4 4.3 13E6 0.19 0.19 12G4 0.34 0.42 Example 6: Competition with HGF for binding to c-Met
Competition with N-terminally biotinylated HGF for binding to immobilized c-Met was performed using an ELISA-based competition test. Five pg / ml of mouse anti-His antibodies (Serotech) were immobilized on a maxisorband plate, after blocking with 1% casein in PBS for 2 h. , 100 ng / ml recombinant dimeric c-Met were added and incubated for 1 h. at room temperature. After washing, dilutions of the antibodies were added and allowed to bind to the captured c-Met for 30 minutes, before 25 ng / ml of N-terminally biotinylated HGF (R&D systems, 294-HGN / CF) was added. Biotinylated HGF was incubated at room temperature for 1 h. before washing. Streptavidin conjugated to horseradish (strep-HRP) was added and incubated for an additional hour. TMB was added and the plate read at 620 nm. An isotype control (hlgGlÀ) was included as a control as well as a murine 5D5 antibody. Competition was expressed as percentage competition as compared to controls (strep-HRP or hlgGlÀ only) and plotted against antibody concentration. An IC50 was calculated using GraphPad Prism (Table 10). Antibodies 13E6 and 20A11 only partially displaced HGF (about 50%), which may be related to the epitope of these two mAbs recognized in c-Met. Figure 6 shows an example of anti-c-Met antibodies competing with HGF for binding to c-Met. Table 10: IC50 of mAbs competing with HGF for binding to c-Met
Example 7: Agonistic and antagonistic properties of mAbs measured in the proliferation test using HGF-dependent pancreatic BxPC3 cells Human pancreatic BxPC3 cells (ATCC cat no.CRL1687) respond to HGF and were used for the proliferation test to further investigate the eight candidate mAbs . In summary, 15,000 cells were seeded in the presence of serum and then left with a serum deficiency overnight following fixation (4 to 6 hours after seeding). The mAbs were added in doses of 20 ng / ml to 40 pg / ml in the presence or absence of 75 ng / ml of HGF in order to test for antagonism and agonism, respectively. After three days of incubation, Alamar blue was added to the cells and incubated at 37 ° C for 4 hours before fluorescence reading with an excitation of 550 nm and emission of 590 nm, thus yielding a reading of the cell proliferation. The test was repeated three times. An example of an experiment performed independently for agonism (Figure 7A) and one for antagonism (Figure 7B is shown for candidate mAbs and reference mAbs including chimeric 224G11 (C224G11, Pierre Fabre). Proliferation is expressed as a percentage of the proliferation obtained with 75 ng / ml HGF Three of the mAbs (38H10, 40B8 and 36C4) showed less than 20% induced proliferation, with 38H10 in the same range as the reference C224G11 Example 8: VL shuffling for improved affinity two mAbs, 38H10 and 48A2. In this method, the parental clone heavy chain (36C4 or 38H10 VHCH1) was reintroduced into the phagemid light chain library (see Example 1). The heavy chain was extracted from an expression vector, which lacked the bacteriophage-derived gene 3 necessary for display, to further prevent contamination of the parental light chain in the selection procedure.The heavy chain was cloned into the phagemid light chain library and the Bound DNA was electroporated into TG1 E.coli cells to create the scrambled light chain library. The size of the libraries was over 108 phages.
The affinity selections, combined with deletion rate washes, to select the scrambled Fabs in the chain with an improved affinity for c-Met. A configuration was chosen where different amounts of phage expressing Fab were incubated with different concentrations of Fc-Met in the solution (see Table 11). By adding excess c-Met on the phage, but at a concentration less than the desired affinity constant, the binding of the highest affinity phage was favored. The Fc-Met: phage complexes were then captured on a microtiter plate coated with an antiFr mAb. The tube was washed with Met decov at 37 ° C to avoid re-binding of the phages dissociated with the captured Fc-Met. In each series, the washing time was increased (see Table 11) to select the phage with a better shutdown rate by washing the lower affinity variants. The phages were eluted with trypsin and used for infection of TGI E. coll. In total, 5 selection series were made. In addition, the amount of phage input was decreased in subsequent series to reduce the bottom on the one hand, and on the other hand to decrease the concentration of mAb thereby increasing the stringency of the selection. Screenings of at least 30 clones from selection series III, IV and V were performed. The clones were cultured in deep well plates (1 ml expressions) and periplasmic fractions were prepared. These periplasmic extracts were first tested for competition with HGF in an ELISA (see Example 2). For 3 8H10 the frequency of the competing clones gave low ELISA signals, increased in subsequent selection series, with the clear enrichment of the competitors in different series.
The clones were then tested for their dissociation constants by surface plasmon resonance. Around 3000 RU of Fc-Met were immobilized directly on a CM5 chip to obtain nm ncvF-ilia 1 i-T arn nart-ir Hna PYt-mice periplasmic. Clones with an improved shutdown rate were sent for sequencing.
Light chains originally from pairs (both Vkappa for 38H10 and Vlambda for 3 6C4) were obtained after 5 shuffling of the light chain, but an improved shutdown rate under the parental Fab was found only for 38H10 variant 48A2 (10-fold Resonance of Surface Plasmon). For 36C4 no improvement in affinity was obtained, so the parental mAb was retained for further 10 jobs. Table 11: Variation of parameter for each VL scrambling selection series.
Several of the Fabs scrambled in the VL sharing the 38H10 variable heavy chain domain (SEQ ID NO: 49). Shuffled light chains are listed below, amino acid and nucleotide sequences are listed in Tables 6 and 7) along with shutdown rates for the corresponding Fabs (each Fab includes 38H10 as the heavy chain) (Table 19).
Several Fabs scrambled in the VL dividing the 36C4Q variable heavy chain domain (SEQ ID NO: 88). The shuffled light chains are listed below (the 10 amino acid and nucleotide sequences are listed in Tables 6 and 7 along with the corresponding Fabs' shutdown rates (each Fab includes 36C4Q as the heavy chain) (Table 20). Table 20
Example 9: Agonistic and antagonistic properties of mAbs measured in the phosphorylation test using HSC-dependent NSCLC A54 9 cells
In order to further investigate the mAbs a phosphorylation test was set up using HSC-dependent NSCLC A54 9 cells (ATCC no. CCL-185). The cells were incubated both in the absence of HGF in order to verify the potent activity of each antibody, as well as in the presence of HGF in order to verify the antagonistic potency of each antibody. In summary, 40,000 cells were plated and left with a serum deficiency overnight after fixing to the plate (after 4-6 h of sowing).
The cells were then treated for 15 minutes at 37 ° C 15 with mAbs. For the antagonism test, 100 ng / ml of HGF was added and incubated for another 15 minutes at 37 ° C. HGF alone (100ng / ml) was also tested to provide reference values for the experiment. The cells were washed with cold PBS and lysed with wild lysis buffer containing PMSF (Cell signaling # 9803 including ImM PMSF, Sigma Aldrich) for 15 minutes on ice. 50 µl of the lysate was added per well in a 96-well plate pre-coated with anti-goat c-Met antibody and blocked with PSB in 1% casein. The c-Met in the lysate was then allowed to bind overnight at 4 ° C. Phospho-c-Met was developed with an anti-rabbit pY1234 / 1235 antibody (cell signaling) and an HRP-conjugated goat anti-rabbit antibody (Jackson Laboratories). TMB was added and the reaction stopped with 1M H2SO4 and read at 450 nm.
The antibodies were tested in duplicate at different concentrations, and the control U16 mAbs (irrelevant mAb, negative control), chimeric 224G11 (c224Gll, Pierre Fabre) and murine 224G11 (mPF, Pierre Fabre) were included in each cycle side by side with HGF only and cells only as positive and negative controls. Figure 8A-B shows the low agonistic effects of the three mAbs as compared to the controls. Compared to the
Reference C224G11, antibodies 38H10, 48A2 and 36C4 phosphorylated. Figure 9 shows the potency of mAbs 48A2, 36C4 and 40B8 in blocking HGF-induced phosphorylation compared to the reference C224G11, with 36C4 having the best blocking power. The percentage of phosphorylation is expressed as the percentage of maximum phosphorylation induced by 100 ng / ml of HGF.
Phosphorylation tests using BxPC3 cells were performed in the same way as for A54 9 cells and the results correlated very well with those obtained with A549 cells (data not shown).
Example 10: Inhibitory effect of anti-cMet antibodies on MKN-45 cells of cMet auto-phosphorylation
To examine the ability of mAbs to inhibit phosphorylation in constitutively activated cells, applicants used gastric MKN-45 cells (DMSZ cat no. ACC 409). These cells have an amplification of the cMet gene resulting in overexpression of c-Met and thus constitutive phosphorylation, that is, independent of HGF.
Briefly, 5,000 cells were seeded in the presence of serum and incubated for 24 h. with different concentrations of mAbs at 37 ° C. An ELISA was performed to quantify the phosphorylated c-Met as described in Example
In Figure 10 the blocking effect of mAbs on phosphorylation of cMet in MKN-45 cells can be seen (% inhibition). The response was normalized against the m16 U16 negative control (0% inhibition). It can be concluded that the 36C4 SIMPLE ™ antibody is the most potent inhibitor of HGF-independent phosphorylation in MKN-45 cells. C224G11 was not as powerful as 3604 and 48A2. 40B8 blocks only around 40% at the highest concentration and speed levels.
Example 11 Antibody-induced ADCC in MKN45 cells. 200,000 MKN-45 cells were seeded the day before the antibody was added. Dilutions of the antibodies were added to the cells and pre-incubated 60 minutes before the effector cells (all PBMCs derived from blood from a donor, incubated overnight before addition to the target cells) were added with an E: T ratio (cells natural killers (NK): target cell lineage) of 5: 1. The subpopulation of the NK cell in PBMCs was determined by flow cytometry for all donors as the ratio of anti-CD16 to anti-CD56. After 4 hours of incubation, the plates were read using the Dead-Cell Protease kit (Promega CytoTox-Glo ™ Cytotoxicity Test
Figure 11 shows the specific lysis induced by the three mAbs, 48A2, 40B8 and 36C4, tested in a dose response compared to C224G11. The EC50 of the three mAbs tested is in the same range as the c224Gll (4.3, 4.6, 5.0, for 48A2, 40B8 and 36C4 and 2.8 ng / ml for C224G11).
Example 12: ADCC induced by 36C4 Potelligent ™ in NCI-H441 cells. Defucosylated 36C4 was produced in CHO Potelligent ™ CHO cells (Biowa) and purified with Protein A. Human donor peripheral blood mononuclear cells (PBMC) from 3 donors were purified separately from all heparinized blood by standard ficoll-type separation were used as effector cells . The cells were suspended in 2x10 * 3 * * 6 * * * 10 / ml in the medium containing 200 U / ml of human IL-2 and incubated overnight at 37 ° C. The following day, adherent and non-adherent cells were collected and washed once in the culture medium.
Target ratios for 1:50 effectors were used. The cells were suspended in 5x10 6 cells / ml and 100 µl added per well. 106 NCI-H441 target cells were incubated with 100 pCi 51Cr in 0.5 ml FCS for 60 minutes in a 37 ° C water bath. The cells were washed, returned to a water bath at 37 ° C. Then the cells were washed twice with the medium and brought to a final volume of 2 x 10 5 cells / ml and 50 µl were added per well.
The test was performed in triplicate. 50 µl of labeled cells were incubated with 100 µl of effector cells and 50 µl of antibody. One row of a 96-well plate contained only target cells to control spontaneous 51 Cr release. On the other 96-well plate, a row of wells contained only target cells treated with 1% Triton-X (in order to completely lyse the cells) giving a maximum release reading of 51 Cr. After 4 hours of incubation at 37 ° C, 50 µl of supernatant was collected, transferred to a 96 well Lumaplate, dried and counted in a beta counter.
The percentage of lysis was determined by the equation:% Lise = ((CPM sample CPM spontaneous release) / (CPM maximum release CPM spontaneous release)) x 100.
Figure 12 shows the percentage of lysis of NCI-H441 cells by 36C4Potelligent ™ (defucosylation-enhanced ADCC) versus normal fucosylated 36C4. Defucosylated 36C4 (36C4Potelligent, IM) induces excellent lysis of NCI-H441 cells with an IC50 of 0.13 nq / ml, whereas normal fucosylated 36C4 does not induce any lysis of NCIH441 cells. The percentage of lysis induced by C224G11 was very low. Clearly the defucosylation of 36C4 dramatically increases its ability to induce ADCC of NCI-H441 cells.
Example 13: In vitro effect of 36C4 with increased ADCC on NCI-H441 cells. mAbs not fucosylated by Potelligent ™ technology have no significant effect in vivo in mice. However, Fc mutations (S239D, I332E) have been shown to have an in vivo effect, increasing the ADCC effect of mAbs by increasing the affinity for mouse FCYRIU, CD16 (Lazar GA et al, PNAS, 103. 2006).
The S239D, I332E mutations were inserted into the 36C4 IgGl using site-directed mutagenesis with specific primers, generating 36C4E. 36C4E was produced in the same way as parenteral 36C4 using HEK293E cells and purified using Protein A. There was no difference in HGF production levels or displacement level in an ELISA-based competition test after mutations compared to parenteral 36C4 . The effect of ADCC was investigated in the 51 Cr release test on NCIH441 cells (as described in Example 12). There was no effect of 36C4 and 36C4 Potelliaent showed slightly less lysis oorcentaaem than Fc mutant 3 6C4E with increased ADCC. The EC5Q for 36C4-POT versus 36C4E was 0.04 pg / ml versus 0.26 pg / ml.
Example 14: In vivo effect of 36C4 with increased ADCC on MKN-45 xenografts.
6-8 week old nude CD-I mice were injected subcutaneously with 3 million MKN-45 cells. The tumors were measurable 8 days after post-injections and treatment was started on day 9 with intraperitoneal injections twice a week with different amounts of the test antibody. Groups of six mice were injected with 36C4E (30, 10, 3 and 1 mg / kg) and the volume of tumors was measured (at the time the injections were performed). An IgGl isotype control (Synagis®) was included as a control as well as C224G11, both at the highest concentration 30 mg / kg.
On day 23 after the injection of the cells (15 days after the start of treatment) a dose-dependent effect on the tumor volume can be observed in the mice treated with 36C4-E. C224G11 had no effect on the tumor as compared to the isotype control (Figure 13).
Example 15: Human chimeric c-Met fusion proteins -Dhama glama p-mt-aínae: Ha fnsãn HP RDC r-Mpt miimárico humann-hlama glama were built by changing the IPT domain of human cMet and Llama glama in order to map the recognition of the domain of mAbs. Construction was done using standard recombinant DNA and PCR methodologies. Human c-Met and Llama glama were amplified from RNA converted to cDNA from peripheral blood lymphocytes (PBLs) from two donors of each species. The llama and human cMet ECD (aa 25-932) were cloned into a eukaryotic expression vector with a His tag for expression as HEK293 cell-soluble proteins. The IPT1-4 (aa 568-932) of the llama was exchanged with the human IPT1-4 in the human c-Met and conversely the IPT1-4 was exchanged with the IPT1-4 llama in the c-Met llama using splice and extension PCR overlap. All four constructs, c-Met llama, llama / human IPT, human c-Met, human IPT / llama were expressed in HEK293 cells and purified using IMAC columns. Figure 15 shows the alignment (88% identity) of the human c-Met (Genbank X54559) with the c-Met Llama glama amplified from the PBLs of two donors.
Example 16: Mapping of mAbs domains using chimeric c-Met ECD. 200 ng of chimeric recombinant c-Met proteins were immobilized on maxisorb plates overnight at
Anns to lavflapm in PR.Q. a.q nlar.as were blocked with 0.1% casein for 2 h in RT, before the mAbs were added and allowed to bind to c-Met for 1 h in RT. After washing, HRP-conjugated goat anti-human antibody (diluted 1/5000, Jackson Labs) was added and incubated for 1 hr in RT before further washing and adding TMB. The optical density at 620 nm was read and the values were plotted against the mAbs concentration.
Figure 16A shows the binding of 36C4 to human c-Met (WT) and human IPT1-4 / llama thus indicating binding to the SEMA-PSI region. Figure 16B shows the binding of 13E6 mAb to human c-Met and to llama / human IPT1-4. No binding was observed for the c-Met llama for any of the mAbs. 48A2 was also tested, but showed mainly a link to the construct with the human SEMA-PSI and some link to the construct with the human IPT, indicating that there was a link to an overlapping region in the PSI-IPT domains.
Example 17: Bonding of 3 6C4 and 4 8A2 for non-overlapping epitopes on c-Met using Surface Plasmon Resonance.
To investigate whether the two 36A4 and 48A2 mAbs linked to the non-overlapping epitopes, 3000 RU of 36C4 or 48A2
Monomeric decoys were injected to form a complex on the chip. 60 pl of 10 pg / ml of 6C4 were injected (Figure 16A). As shown in Figure 16A, binding is observed for the Met: 48A2 complex only. Similarly, binding of mAb 48A2 to the Met: 36C4 complex and Met: 48A2 complex was performed using 3000 RU of 36C4 or 4 8A2 copulated to a CM5 chip. 60 pl 40 pg / ml of Met Decoy were injected to make a complex on the chip. Then 60 µl 10pg / ml 48A2 were injected. Binding was observed for the Met: 36C4 complex only as shown in Figure 16B. These results indicate the recognition of non-overlapping epitopes of mAbs 36C4 and 48A2.
Example 18: Increased inhibitory effect on c-Met autophosphorylation using a combination of anti-cMet antibodies.
The two mAbs 36C4 and 48A2, recognizing non-overlapping epitopes c-Met as shown by Biacore (Figure 16), were combined with a 1: 1 ratio in a phosphorylation test using HGF-independent MKN-45 cells as described in Example 10. The antibody mixture was compared with 36C4 and 48A2 over a range of concentrations for the ability to block autophosphorylation of c-Met (note that the total concentrations of a-nt-n mn open oõn i miai α a rrirr'ejnb rnrãri tribal Hα antibody to the individual antibodies: that is, for the 0.2nM dose the mixture is 0.1nm of each of 36C4 and 48A2, while for pure mAb this could be 0.2nM 36C4 or 48A2) . The combination showed significantly better inhibition of cMet auto-phosphorylation compared to individual mAbs. With 0.78nM mAb, the mixture showed 75% inhibition of phosphorylation compared to 42% and 32% for 36C4 and 48A2 alone (Figure 17). The combination of 36C4 and 48A2 was also more potent than individual antibodies in blocking the auto-phosphorylation of NSCLC EBC-1 cells (data not shown).
Example 19: Combination of non-overlapping mAbs showed lower levels of agonism and better blocking potency in a phosphorylation test using NSCLC A549 cells.
A phosphorylation test using NSCLC A549 cells was performed as in Example 9 to investigate mAbs 36C4 and 48A2 either in combination (1: 1 ratio) or individually for their agonistic and antagonistic activity (in the absence or presence of HGF, respectively). The level of agonism was lower for the combination (3 6C4 and 4 8A2) than for mAbs alone (Figure 18A) and the blocking effect of HGF-induced phosphorylation was significantly increased (36C4 and 48A2) compared to mAbs alone (Figure 18B).
Example 20: Tumor growth inhibition in a U87-MG xenograft model
To avoid the inhibitory effect of mAb 36C4 on tumor growth in vivo, 3x106 U87-MG cells with autocrine HGF (ATCC HTB-14) were injected subcutaneously into the flank of the right rear limb of nude nude CD1 mice. When the tumor reached 70-120 mm3 (day 19), the mice were stratified and treatment started with 30 mg / kg of 36C4 intraperitoneal (i.p.), C224G11 or isotype control antibody twice a week. Treatment continued until day 35 post-injection of the tumor cells, when the experiment was terminated. The tumor size was measured periodically during the experiment when the mAbs were administered and the results are shown in Figure 19. 30 mg / kg of 36C4 inhibits the growth of the U87-MG tumor as well as the comparator mAb C224G11.
Example 21: Germination of 36C4 and 48A2.
The VH and VL sequences of 36C4 and 48A2 were blasted against the VH and human germline sequences and 36C4 was closely related to the germline sequences of IGHV4-30-4 * 01 (66/76 framework identity) and IGLV2- 18 * 02 (61/69 identity of the framework). 4 8A2 was closely related to the germline sequences of IGHV1-46 * O1 (66/76 identity of the framework) and IGKV4-l * 01 (53/70 identity of the framework).
The germinalization process was carried out as described in WO 2010/001251 and by Baca et al. (J. Biol. Chem. (1997) 272: 10678-10684) and Tsurushita et al. (J. Immunol. Methods (2004) 295: 919). It had a library / phage display approach, in which FR waste diverted to both human and llama waste was incorporated. The germline strain library VH36C4 or 4 8A2 and VL36C4 and 4 8A2 was created by the PCR-based gene pool using overlapping oligonucleotides with specific mutations in certain positions (identified in Tables 3 and 4). The mutations were encoded in order to encode the human amino acid as well as the llama, this being to avoid complete loss of binding in the event that the wild type residue is critical for high affinity binding. The gene sets were cloned into a phagemid vector with human CH and CL and TGI E. coli were transformed generating libraries of a total size of 109 clones.
Phage display, applying strict selection conditions (3-5 selection series with decreased amount of antigen and phage and increased length of competitive washes with c-Met access), was used to select functional Fabs (as described in Example 8). Individual clones were screened for shutdown rate and the best hits were sequenced to determine the identity of the human sequence. Clones with> 94% human identity were produced by transient expression when transfecting HEK293E cells and if the yields were> 15 pg / ml, they were characterized again. Table 12: Amino acid sequences of the heavy and light chain domains variables of germinalized variants of lineage 3 6C4

Table 13: Nucleotide sequences encoding variable heavy and light chain domains of 36C4 germline variants


Table 14: Amino acid sequences of the heavy and light chain domains of 48A2 germline variants


Table 15: Nucleotide sequences encoding variable heavy and light chain domains of 48A2 germinalized strain variants



Example 22: Germination of 36C4 did not lead to loss of power. For 36C4, four germline clones (55A12-54E, 53E2-54E, 53E3, 53A11) were again characterized for the agonistic and antagonistic properties in the A549 phosphorylation test as described in Example 9.
As shown in Figure 20A, there were no increased agonistic properties of the mAbs 55A12-54E and 53E2-54E as compared to the parental 36C4. The germline strain 53E3 and 53A11 showed the same results. The antagonistic effect of germinalized lineage mAbs has not been significantly altered or as shown in
Figure 20B, or exemplified by 55A12-54E and 53E2-54E.
Example 23: PBS stability of germinalized lineage 36C4 mAbs.
The stability of 3 mg / ml IgG in PBS + 0.02% Tween-80 was investigated on days 0-1-7-14-28-56 after storage at 4 ° C, RT and 37 ° C. All samples were tested for their power by Surface Plasmon Resonance investigating the binding to the copulated c-Met (15,000-17,000 RU) and determining deviation between 100-130 seconds with a flow rate of 30 pl / min. The percentage of functional mAbs was calculated based on the reference (germinalized mAbs at -20 ° C). Figure 21 showed that there was no significant loss of functionality after 56 days of incubation at different temperatures and there did not appear to be a significant difference between the four mAbs of germline lineage.
Example 24: Thermotolerance of germinal lineage 36C4 and 48A2 mAbs
The thermotolerance of mAbs 3 6C4 and 4 8A2 of germ line was investigated by incubation at different temperatures for 1 h before the samples (0.5 pg / ml) were worked on a CM-5 chip copulated with 15,000-17,000 RU of c -Met Decoy and a decline determining the decline between 100-130 seconds with a flow rate of 30 ul / min. Dorcentaaem of functional mAbs was calculated based on the reference (incubated at 4 ° C) adjusted to 100%.
As shown in Figure 22A, the melting temperatures (EC50) of the germline lineage mAbs was 67.2 ° C for 36C4, 67.1 ° C for 55A12-54E, 66.1 ° C for 53E2-54E, 68.2 ° C for 53E3 and 65.5 ° C for 53A11. For 48A2, the mAb 56F3 of germline lineage, had a significant improvement in the melting temperature from 65.4 to 71.1 ° C (Figure 22B).
Example 25: Determination of the c-Met peptide binding sites of mAbs 36C4 and 48A2 using human chimera / llama chimeric c-Met
To further define the extent of the amino acids (aa) of the c-Met to which mAbs 36C4 and 48A2 were attached, chimeric c-Met constructs containing exchanges of approximately 20300 aa of human c-Met for llama were prepared using PCR amplifications and bonds inside the human c-Met containing the vector with a flag and a strep tag. Figure 23A shows the chimeric c-Met constructs used for mapping the 36C4 binding peptide to the SEMA domain, while Figure 23B shows the chimeric c-Met constructs for mapping the 48A2 binding peptide to the PSI domain. -IPT1.
The llama-human c-Met chimeras were produced in HEK293E cells and purified using strep tactin sepharose HP (2-3 h at 11 ° C) before washing the unbound proteins. Bound proteins were eluted with 2.5 mM destiobiotin pH 8.2 and 1.5 ml fractions were collected. The protein concentration was determined by Nanodrop. The quality of the protein was controlled by SDSPAGE.
An ELISA was performed to investigate the binding of mAbs to different chimeras. 2 pg / ml of 36C4 or 48A2 were immobilized and, after blocking, c-Met chimeras were added and developed with 1 / 10,000 streptavidinHRP (ELISA in Table 16).
Surface Plasmon Resonance (SPR) was also used to investigate the binding of mAbs to chimeras other than llama-human c-Met. 3000 RU of 36C4, 48A2 and HGF were copulated on a CM-5 chip of 10 mM NaAc (pH4.5). 60 pl of a 10 pg / ml solution that chimeras other than c-Met were worked over the chip with a flow rate of 30 pl / min and the association was evaluated for 2 min. The chip was regenerated with 20 mM NaOH and 1 M NaCl.
Table 16 shows the chimeras with human c-Met and the amino acids (starting with aa E in the mature human c-Met protein) that were exchanged with the c-Met llama peptides and the binding results using Plasmon Resonance and linkage of 3 6C4 to no aa 199, indicating a recognition site within aa 98-199 of human c-Met. This is the part of the SEMA domain that contains the β chain binding site for HGF, as shown in the crystal structure published by Stamos et al, (EMBO J, 2004).
The mAb 48A2 linked to aa 523-633 of the human c-Met, which covers both parts of the PSI and IPT1 domains, indicates the recognition of a conformational epitope in both domains.
Western Blot with c-Met operating under reduced conditions was used to investigate whether 36C4 and 48A2 bound to linear or conformational epitopes. No binding was observed for 36C4 or 48A2 indicating recognition of a conformational epitope (data not shown), which was confirmed with the chimeric c-Met proteins.
Table 16: Chimeras of the llama-human c-Met and binding results of 36C4 and 48A2 measured by SPR and ELISA


Human c-Met peptide sequence recognized by mAb 36C4 (aa98-199) SEQ ID NO.-181 VDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDC WSALGAKVLSSVKDRFINFFVGNTINSSYVPNKIN
Human c-Met peptide sequence recognized by mAb 48A2 (aa523-633) SEQ ID NO: 136 RVERCLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKK TRVLLGNESCTLTLSESTMNTLKTTVGAMN
Example 26: Negative regulation of total c-Met by mAbs in MKN-45 cells
The total amount of cMet present on the surface of MKN-45 cells after incubation with the mAbs was measured using flow cytometry. 25,000 MKN-45 cells / well in a 96-well plate were seeded and incubated for 24h at 37 ° C, 5% CO2. The cells were left with a serum deficiency for 8 h before the addition of mAbs and HGF with 10 or 1 pg / ml diluted in serum-free medium and in triplicates. The murine 5D5 antibody and HGF were included as controls for negative regulation of total c-Met. The negative control is an irrelevant IgGl produced in the same way as 36C4 and 48A2.
The cells were washed with PBS and 50 µl / well of the cell dissociation solution was added and incubated for 15 min at 37 ° C. The cells were collected on a FACS plate and 100 µl of binding buffer (PBS + 1% BSA) was added before centrifugation at 2000 rpm for 3 min. The cells were maintained at 4 ° C from this point on. The cells were washed twice with binding buffer and then 2.5 pg / ml of anti-rat c-Met antibody (R&D systems) added. The cells were then incubated for 1 h with shaking at 4 ° C, followed by two washes with the binding buffer. APC-conjugated goat anti-mouse antibody (Jackson Lab) was added at a concentration of 1/500 and the cells incubated for 1 h with shaking. The cells were then washed with binding buffer and read on a FACS Calibur. 2000 events and negative regulation were expressed as a percentage of negative regulation in the control of the medium.
Figure 24 shows that mAbs 36C4 and 48A2 do not induce significant negative regulation of c-Met on the surface of MKN-45 cells compared to 5D5 or HGF, both of which induce 50-60% of negative regulation of cMet after overnight incubation. Example 27; Agonistic properties of mAbs combinations measured in a phosphorylation test using HGF-dependent NSCLC A54 9 cells
In order to further investigate the agonistic properties by combining two c-Met mAbs, a phosphorylation test was configured using HGF-dependent NSCLC A549 cells (ATCC no. CCL-185). This test was performed in the absence of the HGF in order to check the agonist activity of each antibody test reagent. 40,000 cells were plated and left with a serum deficiency after fixing to the plate (4-6 h after sowing). The cells were then treated for 15 minutes at 37 ° C with mAbs. HGF alone (100ng / ml) was also tested to provide reference values for the experiment. The cells were washed with cold PBS and lysed with wild lysis buffer containing PMSF (Cell signaling # 9803 including 1 mM PMSF, Sigma Aldrich) for 15 minutes on ice. 50 µl of lysate was added per well in a 96-well plate pre-coated with goat anti-cMet antibody blocked with 1% casein-PSB and the c-Met in the lysate was allowed to bind overnight at 4 ° C . Phospho c-
Met was developed with an anti-rabbit pY1234 / 1235 antibody (cell signaling) and an HRP-conjugated goat anti-rabbit antibody (Jackson Laboratories). TMB was added and the reaction stopped with 1M H2SO4 and read at 450 nm.
The antibody combinations were tested in duplicates at different concentrations, and the mAbs alone as single samples. The control U16 (negative control, irrelevant mAb) and chimeric 224G11 (c224Gll, Pierre Fabre) mAbs were included in each cycle as well as HGF and a cell-only background control. Figure 26A-C shows the agonistic effects of the different combinations of mab. Figure 26A shows the agonistic effects when combining two mAbs binding to non-overlapping epitopes in the SEMA domain compared to the mAbs alone and the combination of 36C4 and 48A2, where 36C4 binds to the SEMA domain and 48A2 to the IPT. The combination of the two SEMA ligands shows a significantly lower level of agonism compared to the individual mAbs tested alone.
Figure 26B shows that the combination of a SEMA ligand (36C4 or 34H7) and an IPT ligand (48A2 or 13E6) can give significantly different agonistic responses when combined. The combination of 36C4 and 48A2 is significantly less agonistic than the combination of 34H7 and 13E6. IPT, 13E6 and 48A2 compared to the combination of 36C4 and 48A2. Again, the 36C4 / 48A2 combination showed a lower level of agonism than the 13E6 / 48A2 combination, which surprisingly showed higher levels of agonism when combined then when added alone. The percentage of phosphorylation is expressed as the percentage of maximum phosphorylation induced by 100 ng / ml of HGF. Example 28: Antagonistic effects of mAb combinations on self-phosphorylated MKN-45 cells
To examine the ability of mAbs to inhibit phosphorylation in constitutively activated cells, applicants used gastric MKN-45 cells (DMSZ cat no. ACC 409). These cells have an amplification of the c-Met gene resulting in overexpression of c-Met and thus constitutive phosphorylation, that is, independent of HGF. 5,000 cells were seeded in the presence of serum and incubated for 24 h with different concentrations of the combinations of mAbs (800 nM means 400 nM of each mAb) or mAbs alone at 37 ° C. An ELISA was performed to quantify the phosphorylated c-Met as described in the example for A549 cells. In Figure 27A-C the blocking effect of mAb combinations tested at various concentrations on phosphorylation of cMet (% inhibition) in MKN-45 cells can be seen. The response was normalized against the u16.1 mAb negative control (0% inhibition). It can be concluded that all combinations of mAbs showed inhibitory effects of phosphorylation in MKN-45 cells.
Figure 27A shows the inhibitory effects of the combination of two mAbs binding to non-overlapping epitopes in the SEMA domain, reaching 80% at 800 nM, which is as potent as the combination of 36C4 and 48A2. In figure 27B, the combination of 36C4 and 48A2 is more effective in blocking phosphorylation than the other combination of a SEMA and an IPT ligand (20F1 and 13E6). In Figure 27C, two IPT ligands inhibit phosphorylation better than individual mAbs alone, but not to the same extent as the 36C4 / 48A2 combination. C224G11 was not as potent as the combination of 36C4 and 48A2. 40B8 blocks only around 40% at the highest concentration and speed levels. Example 29: Scattering test using HPAF cells
Human pancreatic cancer cells with serum deficiency (HPAF) were plated in 96 well plates, 7000 cells / well. On day 2, antibodies were added in triplicates at concentrations of 30 (15 + 15 nars A mnhinarãnl. 10 3 n 1 water / ml and incubated with the cells for 30 minutes before 40 ng / ml HGF / well was added. HPAF cells were also incubated with antibodies in the absence of HGF, and on day 3, the cells were fixed and stained with violet crystal. The scattering quantity score was done three times independently and by two different people.
The results in figure 28 show that the blocking effect of the 36C4 and 48A2 combination is 10 times as good at blocking the spreading induced by HGF compared to the individual mAbs alone. No agonistic properties were observed. No other combination (36C4 / 13E6, 48A2 / 13E6, 36C4 / 20F1, 48A2 / 20F1) investigated was as potent in blocking as the combination of 36C4 and 48A2. Example 30 Transient expression of camelid-derived bi-specific c-Met antibodies
Camelid-derived antibody antibodies are generally expressed at very high levels (> 20 µg / ml in transient transfections of HEK293E cells). In addition, during the selection of functional Fabs, families of VH pairs with the same VL are generally isolated, leading applicants to believe that both VH and VL are involved in binding to the epitope. This discovery is rααiil t-aHnc scrambling of the VL where high affinity, functional Fabs are selected to, generally reveal only the original VL variant. Based on these properties of SIMPLE Antibodies, the applicants concluded that by coexpressing two different antibodies (the first VH1 / VÜ and the second VH2 / VD) relatively high levels of bi-specific antibodies with correct pairs of VH1 / V0 and VH2 / VD can be formed.
Applicants investigated whether forced expression of the VH and VL chains placed in pairs incorrectly would yield mAbs (protein level) and also determined their functionality in epitope binding studies.
To synthesize bi-specific antibodies of the invention, a panel of anti-c-MET, camelid-derived, monoclonal antibodies having binding sites placed in VÀ / VH or VK / VH pairs that recognize different domains of the c-Met target (see Table 17), were used.
Antibodies encoding plasmid with VÀ / VH and VK / VH binding sites were mixed in the following relationships. 1 = plasmid ratio 36C4: 40B8 1: 1 2 = plasmid ratio 36C4: 38H10 1: 1 3 = plasmid ratio 20F1.-4 0B8 1: 1 4 = plasmid ratio 20Fl: 38H10 1: 1 5 = ratio of plasmid 36C4: 40B8 2: 1 6 = ratio of plasmid 36C4: 38H10 2: 1 50 ml HEK293E cells with a total of 25 pg of plasmid mix and the mAbs were produced for 6 days before purification of the mAb with Protein beads A. After purification a mixture of parental mAbs and specific mAbs was obtained.
An ELISA was configured according to the schematic illustration in Figure 29. SEMA-PSI were coated and after blocking with casein, mAbs were added (samples 1-6) in dilutions as well as controls for parental mAbs. After 1 h of incubation and washing, both mouse anti-human CK and HRP-conjugated goat anti-human FK were added and incubated for another hour. Mouse CKanti-human was detected with an HRP-conjugated anti-mouse mouse antibody. This test will reveal only the bound bispecific antibody, since parental antibodies containing lambda can recognize immobilized SEMA-PSI and parental antibodies containing kappa cannot. On the other hand, by using the goat anti-human Fc antibody instead of the anti-human C kappa antibody, all combinations of functional mAbs binding to SEMA-PSI (parental and bi-specific) (Figure 30). To recap, bi-specific mAbs that bind with a first arm (comprising the 36C4 or 20F1 VÀ / VH binding site) to SEMA-PSI and with the second arm (comprising the 40B8 or 38H10 VK / VH binding site) are specifically detected with the mouse CKanti-human antibody (Figure 31) that binds to a CK domain fused to the VK domain. Results
After applying the culture supernatant to the protein A columns, between 0.5-2 mg of the mAbs were purified, which is the normal production range for parenteral mAbs. SEMA-specific mbAbs 36C4 and 20F1 containing a VÀ / VH binding site were produced in the purified antibody mixture with protein A as shown in Figure 30, as binding could be demonstrated with the anti-human Fc antibody. As expected, parenteral antibodies 36C4 and 20F1 specifically bound to SEMA-PSI, but not to parenteral antibodies 38H10 or 40B8, which are of specific IPT.
In Figure 31 the mixtures of purified antibodies were tested for the presence of the bi-specific antibody using the ELISA configuration in Figure 29. Bi-specific mAbs were produced by mixing 36C4 or with plasmids 38H10 or 40B8 for transfection as can be seen in Figure 3, where the The 36C4 VÀ / VH binding site is binding to the SEMA-PSI domain and the 38H10 or 40B8 VK / VH binding site is binding to the IPT domain. These antibodies were detected with the anti-human CK antibody that binds to a CK domain fused to the VK / VH binding site domain. No binding was observed for mono-specific parental mAbs 40B8 or 38H10 or for secondary antibodies, thus validating the test to demonstrate bi-specific binding. Although bi-specific antibodies have been produced from mixtures of 20Fl: 38H10 and 2OF1: 4OB8 at lower levels, these could also be detected in a bi-specific ELISA. Example 31; Expression and purification of bi-specific cMET antibodies, derived from camelid
To facilitate the purification of bi-specific cMET antibodies, derived from camelid, a three-step column purification process was employed. First, the antibodies were purified on a ProtA sepharose column to select only the Mabs in appropriate sets, containing two heavy and two light chains. A fraction of the purified antibody was purified again, first in Selection-Lambda accounts and then in Selection-Kappa accounts (BAC BV), thereby separating the parent Mabs from the specific Mabs. The following mixtures with the "wrong" combinations (i.e., binding sites without VH / VÀ and VH / VK pairs containing promiscuous VÀ or VK light chains) were made for transfections on a 20 ml scale: 1 = VH36C4 ratio of plasmid : VK40B8 1: 1 2 = plasmid ratio VH40B8: VL36C4 1: 1 3 = plasmid ratio VH36C4: VK38H10 1: 1 4 = plasmid ratio VH38H10: VL36C4 1: 1 Bi specific functional (ie antibodies with binding sites VH / VÀ and VH / VK appropriately placed in pairs containing VÀ or VK light chains that contribute to the antigen binding function of the binding site) were obtained by transfections on a 200 ml scale using the following combinations of plasmids: 5 = Plasmid ratio VHVL36C4: VHVK40B8 1: 1: 1: 1 6 = Plasmid ratio VHVL36C4: VHVK38H10 1: 1: 1: 1 T amph-o iini2 L108Q in the heavy chain (SEQ ID: 88) was used here. This mutant was found to be more highly expressed than its wild type or Mab. In fact, the expression levels of this variant are comparable to the expression levels of 40B8 and Mabs 38H10. Results
Cultures of HEK293E cells were transfected with plasmid mixtures encoding HC and LC of 36C4 and 38H10 / 40B8, respectively, or with forced wrong combinations of the VH and VL of these mAbs. Following transfection, the culture of supernatants was collected and purified on sepharose beads in protein A. Subsequently the antibody preparation was further purified on Selection-Lambda beads or Selection-Kappa beads for cultures expressing the forced wrong combinations of VH and VL (transfection 1 to 4), while antibody fractions for bi-specific antibodies (transfection 5 and 6) were purified first in Selection-Lambda beads subsequently in Selection-Kappa beads. The yields of the purification steps are shown in Table 18. Table 18. Production yields of transiently transfected HEK293E cells expressing bi-specific anti-cMet antibodies and forced wrong combinations of VH and VL

Purification samples (flow through the protein A column and the purified Selection-Kappa and / or 5 Selection-Lambda fractions) were analyzed in Coomassie Brilliant Blue (CBB) colored gels or under reduction conditions, that is, boiled in a sample buffer containing DTT (Figure 32A), or under non-reduction conditions without DTT (Figure 32B).
Really large amounts of antibody were produced and purified from cultured cells transfected with the wrong forced combinations of VH and VL (transfection 1 to 4). Purification of Protein A followed by Selection-Kappa or Selection-Lambda revealed that these 15 "unpaired" binding sites form an appropriate antibody with both heavy and light chains, suggesting that unmatched light chain forms exist in the population. In particular, the fraction through the flow of the strong combination reinforced with VL36C4 (numbers 2 and 4) appeared to contain a free heavy chain (Figure 32A), while in the unreduced sample an additional band appeared to be migrating to the higher band of the marker (Figure 32B).
The specific bifunctional fractions (samples 5 and 6) were found to contain a mixture of light chains as can be clearly seen in the gel with the reduced samples (Figure 32A). The purified fractions from all transfected cultures were tested in the bi-specific ELISA of Figure 29 using the immobilized SEMA domain and the CKanti-human antibody for detection (Figure 33A). In parallel, the fractions were tested in the same ELISA, but using the anti-human Fc antibody to detect both parental and bi-specific formats of the 36C4 antibody (Figure 33B). In contrast to bi-specific 38H10 and 40B8 antibodies, forced "wrong" combinations of VH and VL (transfection 1 to 4) may not bind to the coated SEMA domain (Figure 33). For this reason, although the wrong combinations of VH and VL can form an antibody with both heavy and light chains, they do not appear to form an appropriate paratype for binding to the SEMA domain. Thus, "peerless" combinations do not form a functional binding site, indicating that both the VH and VL domains contribute to binding.
The ELISA shown in Figure 34 revealed that each purification step enriched the bispecific antibodies by removing the parental antibodies. Consequently, it can be concluded that during the purifications in Selection-Kappa and Selection-Lambda the produced bi-specific mAbs can be successfully separated from the parental mAbs, but that probably some unique antibody combinations were co-purified. Discussion
The examples describe the generation of bi-specific constructs containing both camelid-derived VH / VK and VH / VÀ binding sites recognizing different domains (SEMA versus IPT) of the cMET receptor. Transfection of HEK293 cells was performed with plasmid mixtures encoding the VH and VL of two cMet antibodies and various combinations of SIMPLE antibodies were generated. The presence of bi-specific antibodies in the culture of supernatants from the transfected cells was demonstrated using a dedicated ELISA, in which SEMA binding was detected for antibodies containing VH / VÀ and detection was performed with the anti-human CK antibody recognizing the antibodies SIMPLE specific IPT. In fact, unexpectedly high levels of bi-specific antibodies have also been produced. Without wishing to be limited by any particular theory, it is believed that the high levels of expression of parental antibodies allowed the production of high amounts of bi-specific antibodies.
Although "peerless" bi-specific antibodies have been produced, it should be emphasized that not a single antibody with a forced wrong VH-VL combination can bind to SEMA thus demonstrating the importance of the camelid-derived antibody light chain in interacting with the antigen. In addition, the subsequent purification in SelectionKappa and Selection-Lambda also showed higher concentrations of the bi-specific antibody as was concluded based on the higher signals in the bi-specific ELISA. In the CBB-colored gel the antibody actually appeared to have two different light chains.
Decades ago, it was suggested to apply two antigen-based affinity purification columns in sequential order to eliminate the two parental antibodies, the formats with an antigen binding arm and all non-functional combinations, thus yielding the bi-specific antibody in the completely purified form. Since the majority of the antibodies in the mixture are eliminated in this purification approach, it is important to have very good levels of expression of the antibodies (as seen with the anti-c-Met anti-c-Met choroids described here) as well as a costly purification method effective in order to have a viable process.
The preferred solution may be to use anti-idiotypic antibodies or antibody fragments that specifically recognize the functional antibody for sequential purifications. The application of monovalent Fab fragments should be preferred over the full-length bivalent IgG format, as it allows for a less stringent solution during affinity purification.
In conclusion, the extremely good expression levels of antibodies derived from camelids overcome the production problems observed for hybrid hybridomas.

权利要求:
Claims (16)
[0001]
1. Isolated monoclonal antibody, or antigen-binding fragment thereof, characterized by the fact that it specifically binds to human c-Met protein, said antibody or antigen-binding fragment comprising a variable heavy chain domain comprising CDR3, CDR2 and CDR1, and a variable light chain domain comprising CDR3, CDR2 and CDR1, wherein: the sequence of variable heavy chain CDR3 is SEQ ID NO: 21; the variable heavy chain CDR2 sequence is selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 83 and SEQ ID NO: 84; and the variable heavy chain CDR1 sequence is SEQ ID NO: 19; and the variable light chain domain includes a combination of CDRs selected from the following: (i) the variable light chain CDR3 sequence is SEQ ID NO: 33; the variable light chain CDR2 sequence is SEQ ID NO: 32; the variable light chain CDR1 sequence is SEQ ID NO: 31; or (ii) the variable light chain CDR3 sequence is SEQ ID NO: 145; the variable light chain CDR2 sequence is SEQ ID NO: 32; the variable light chain CDR1 sequence is SEQ ID NO: 14 4; or (iii) the variable light chain CDR3 sequence is SEQ ID NO: 146; the variable light chain CDR2 sequence is SEQ ID NO: 32; the variable light chain CDR1 sequence is SEQ ID NO: 31; or (iv) the variable light chain CDR3 sequence is SEQ ID NO: 147; the variable light chain CDR2 sequence is SEQ ID NO: 32; the variable light chain CDR1 sequence is SEQ ID NO: 14 4; or (v) the variable light chain CDR3 sequence is SEQ ID NO: 148; the variable light chain CDR2 sequence is SEQ ID NO: 32; the variable light chain CDR1 sequence is SEQ ID NO: 144.
[0002]
2. Isolated monoclonal antibody, or antigen-binding fragment thereof, according to claim 1, characterized by the fact that: the variable heavy chain CDR3 sequence is SEQ ID NO: 21; the variable heavy chain CDR2 sequence is SEQ ID NO: 83; the variable heavy chain CDR1 sequence is SEQ ID NO: 19; and the variable light chain comprises CDR3, CDR2 and CDR1, wherein: the CDR3 sequence of the variable light chain is SEQ ID NO: 33; the CDR2 sequence of the variable light chain is SEQ ID NO: 32; and the CDR1 sequence of the variable light chain is SEQ ID NO: 31.
[0003]
3. Isolated monoclonal antibody, or antigen-binding fragment thereof, according to claim 2, characterized by the fact that the variable heavy chain (VH) domain comprises the amino acid sequence shown as SEQ ID NO: 94, and that the variable light chain (VL) domain comprises the amino acid sequence shown as SEQ ID NO: 95.
[0004]
4. Antigen or antigen-binding fragment according to any one of claims 1 to 3, characterized by the fact that it exhibits one or more effector functions of the antibody selected from antibody-dependent cell-mediated cytotoxicity (ADCC), dependent cytotoxicity complement (CDC) and antibody dependent cell mediated phagocytosis (ADCP) against cells expressing human c-Met protein on the cell surface.
[0005]
Antibody or antigen-binding fragment according to claim 6, characterized by the fact that it comprises an Fc domain that is modified in relation to a native human Fc domain and that exhibits marked effector function compared to a reference antibody with substantially identical antigen binding specificity, but comprising a native human Fc domain, by at least one effector function selected from antibody dependent cell mediated cytotoxicity (ADCC).
[0006]
An antigen-binding antibody or fragment according to any one of claims 1 to 5, characterized in that it contains the fold region, CH2 domain and CH3 domain of a human IgG, preferably IgG1.
[0007]
Antibody according to any one of claims 1 to 6, characterized in that it is a partially or entirely defucosylated IgG.
[0008]
8. Isolated polynucleotide characterized by the fact that it encodes the antibody or antigen binding fragment as defined in any of claims 1 to 7 and comprises at least one of the following sequence pairs: SEQ ID: 59 and 65, SEQ ID NO: 100 and 101, SEQ ID NO: 102 and 103, SEQ ID NO: 104 and 105 and / or SEQ ID NO: 106 and 107.
[0009]
9. Expression vector characterized by the fact that it comprises the polynucleotide as defined in claim 8 operably linked to regulatory sequences that allow the expression of the antigen-binding polypeptide in a host cell expression system or a cell-free expression system.
[0010]
10. Expression system characterized by the fact that it comprises the expression vector as defined in claim 9.
[0011]
11. Method for producing a recombinant antibody or antigen-binding fragment thereof comprising the culture of a host cell expression system, or cell-free expression system as defined in claim 10, under conditions that allow expression of the antibody, or its antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment, as defined in any one of claims 1 to 7.
[0012]
Pharmaceutical composition characterized in that it comprises an antibody or antigen-binding fragment as defined in any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient.
[0013]
13. Immunoconjugate characterized by the fact that it comprises an antibody or antigen-binding fragment as defined in any one of claims 1 to 7 and a cytotoxic agent, cytostatic agent, toxin or radionuclide.
[0014]
14. Use of an antibody or antigen-binding fragment as defined in any one of claims 1 to 7 characterized by the fact that it is for the production of a cancer treatment drug.
[0015]
15. Use of an immunoconjugate as defined in claim 13, characterized by the fact that it is for the production of a cancer treatment drug.
[0016]
16. Use according to claim 14 or 15, characterized by the fact that the therapeutically effective amount of the drug is between about 0.1 mg / kg to about 20 mg / kg of the patient's body weight.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-02-27| B25F| Entry of change of name and/or headquarter and transfer of application, patent and certif. of addition of invention: change of name on requirement|Owner name: ARGEN-X BV (NL) |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2018-06-26| B25D| Requested change of name of applicant approved|Owner name: ARGEN-X N.V. (NL) |

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2018-07-10| B25D| Requested change of name of applicant approved|Owner name: ARGENX SE (NL) |

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2018-08-21| B25A| Requested transfer of rights approved|Owner name: ARGENX BVBA (BE) |

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2019-05-21| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-04-14| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-08-11| B09A| Decision: intention to grant|

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2020-11-17| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/11/2011, 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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US40986610P| true| 2010-11-03|2010-11-03|

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US61/409,866|2010-11-03|

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PCT/EP2011/069369|WO2012059561A1|2010-11-03|2011-11-03|Anti c-met antibodies|BR122014027420-3A| BR122014027420A2|2010-11-03|2011-11-03|ANTI C-MET ANTIBODIES|