An antibody or antigen-binding fragment thereof that specifically binds to human cMET

Abstract

The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human cMET. The present disclosure also provides polynucleotides encoding the antibody or antigen-binding fragment thereof, vectors comprising the polynucleotides, host cells comprising the vectors, methods of making the antibody, and compositions comprising the antibody.

Description

Technical Field

[0001] This disclosure provides an antibody that specifically binds to human cMET, or an antigen-binding fragment thereof. This disclosure also provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof, a vector comprising the polynucleotide, a host cell comprising the vector, a method for generating the antibody, and a composition comprising the antibody. Background Technology

[0002] Receptor tyrosine kinase mesenchymal-epithelial transformation factor (c-MET, also known as cMET or MET, hepatocyte growth factor receptor, or HGFR) and its ligand hepatocyte growth factor (HGF) play a key role in stimulating multiple cellular processes such as cell proliferation, survival, invasion, and angiogenesis.

[0003] cMET consists of an extracellular α subunit and a transmembrane β subunit linked by disulfide bonds. The extracellular region comprises a brain signaling protein (SEMA) domain, a panicin-brain signaling protein-integrin (PSI) domain, and four consecutive immunoglobulin-panunclin-transcription factor (IPT1-4) domains. HGF binding to cMET induces cMET dimerization, leading to autophosphorylation of its intracellular kinase domain (KD). This, in turn, creates active docking sites for proteins that mediate activation of downstream signaling pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT pathways.

[0004] Dysregulation of HGF-cMET signaling has been observed in various solid cancer types, including gastric cancer, colorectal cancer, lung cancer, liver cancer, head and neck cancer, breast cancer, and brain cancer. Aberrant cMET activation in an HGF-independent manner can be induced by multiple mechanisms, including cMET overexpression, genomic amplification, mutation, and alternative splicing. Given its important role in cellular processes, tumorigenesis, and cancer progression, c-MET is considered a promising target for cancer therapy.

[0005] There remains an unmet medical need for targeted therapies for cMET. Summary of the Invention

[0006] This disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human cMET, which meets the needs of the art.

[0007] On one hand, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human cMET, comprising: a heavy chain variable region (VH), the heavy chain variable region comprising CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, and CDR-H3 as shown in SEQ ID NO: 9; and a light chain variable region (VL), the light chain variable region comprising CDR-L1 as shown in SEQ ID NO: 12, CDR-L2 as shown in SEQ ID NO: 13, and CDR-L3 as shown in SEQ ID NO: 14.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises: VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO: 10; or VH having at least 95%, 96%, 97%, 98%, or 99% sequence identity with VH shown in SEQ ID NO: 5 and VL having at least 95%, 96%, 97%, 98%, or 99% sequence identity with VL shown in SEQ ID NO: 10.

[0009] In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises CDR-H1, CDR-H2 and CDR-H3 from VH as shown in SEQ ID NO: 5, and CDR-L1, CDR-L2 and CDR-L3 from VL as shown in SEQ ID NO: 10, for example according to the definition of Kabat, Chothia or IMGT.

[0010] In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises a heavy chain constant region as shown in SEQ ID NO: 15 and a light chain constant region as shown in SEQ ID NO: 16.

[0011] In one embodiment, the disclosed antibody or its antigen-binding fragment comprises an α heavy chain, a δ heavy chain, an ε heavy chain, a γ heavy chain, or a μ heavy chain. In one embodiment, the disclosed antibody or its antigen-binding fragment comprises a heavy chain constant region derived from an IgG1, IgG2, IgG3, or IgG4 subclass. In one embodiment, the disclosed antibody or its antigen-binding fragment comprises a light chain constant region derived from a λ light chain or a κ light chain. In one embodiment, the disclosed antibody or its antigen-binding fragment is a full-length antibody. In one embodiment, the disclosed antibody or its antigen-binding fragment is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, and xFab. In one embodiment, the disclosed antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody or its antigen-binding fragment. In one embodiment, the disclosed antibody or its antigen-binding fragment is a multispecific antibody, such as a bispecific antibody.

[0012] In one embodiment, the disclosed antibody or its antigen-binding fragment has one or more of the following properties: (1) it can specifically bind to human cMET protein; (2) it can specifically bind to cells expressing human cMET protein; (3) it can specifically bind to cyno cMET protein; (4) it can specifically bind to cells expressing cyno cMET protein; (5) It can inhibit HGF-cMET interaction and suppress its downstream signal transduction activity.

[0013] On one hand, this disclosure provides a polynucleotide encoding an antibody or antigen-binding fragment thereof. In one embodiment, the polynucleotide is codon-optimized to suit expression in mammals.

[0014] In one embodiment, the polynucleotide disclosed herein comprises: SEQ ID NO: 6 and / or 11.

[0015] On one hand, this disclosure provides a vector comprising the polynucleotides of this disclosure. In one embodiment, the vector is a plasmid, granule, bacteriophage, phage particle, or virus.

[0016] On one hand, this disclosure provides a host cell comprising the polynucleotides or vectors disclosed herein. In one embodiment, the host cell is a eukaryotic cell. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is *Escherichia coli*.

[0017] On one hand, this disclosure provides a method for generating an antibody or an antigen-binding fragment thereof, comprising: (a) culturing a host cell of this disclosure under conditions suitable for expressing the antibody or the antigen-binding fragment thereof, and (b) optionally, recovering the antibody or the antigen-binding fragment thereof.

[0018] On the one hand, this disclosure provides a composition comprising an antibody of this disclosure or an antigen-binding fragment thereof, a polynucleotide of this disclosure, a vector of this disclosure, or a host cell of this disclosure. Detailed Implementation

[0019] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Otherwise, certain terms used herein have the meanings set forth in the specification.

[0020] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a”, “an”, and “the” used herein and in the appended claims include plural indicators.

[0021] Unless otherwise stated, any numerical value (such as concentrations or concentration ranges as described herein) should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​typically include ±10% of the listed values. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context explicitly specifies otherwise, as used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions of such values.

[0022] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of this disclosure described herein using only conventional experimentation. Such equivalents are intended to be covered by this disclosure.

[0023] As used herein, the terms “comprising,” “including,” “having,” or “containing,” or any other variation thereof, shall be understood to imply inclusion of the integers or groups of integers stated therein, but not to exclude any other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, “or” means inclusive or rather than exclusive or. For example, conditions A or B satisfy any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0024] As used herein, the connecting term "and / or" between multiple enumerated elements is understood to encompass both individual and combined options. For example, in the case where two elements are connected by "and / or," the first option means that the first element applies while the second element does not. The second option means that the second element applies while the first element does not. The third option means that both the first and second elements apply. Any of these options is understood to fall within the aforementioned meaning and thus satisfy the requirement of the term "and / or" as used herein. The simultaneous application of more than one of the options is also understood to fall within the aforementioned meaning and thus satisfy the requirement of the term "and / or".

[0025] As used herein, the term "consisting of" or its variations as used throughout the specification and claims indicates that it includes any of the enumerated integers or groups of integers, but no other integers or groups of integers may be added to the specified method, structure, or composition.

[0026] As used herein, the term “consistently of” or its variations as used throughout the specification and claims indicates that it includes any enumerated integers or groups of integers, and optionally includes any enumerated integers or groups of integers that do not substantially alter the essential or novel characteristics of the specified method, structure, or composition.

[0027] As used herein, “subject” or “individual” means any animal, preferably a mammal, and most preferably a human. The term “mammal” as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with a preference for humans.

[0028] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and similar terms used herein, when referring to the size or feature of a component of a preferred invention, indicate that the described size / feature is not a strict boundary or parameter and does not exclude minor variations that are functionally identical or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations that do not change the least significant figure using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0029] In the context of two or more nucleic acid or polypeptide sequences (e.g., anti-cMET antibody and the polynucleotide encoding them, cMET polypeptide and the cMET polynucleotide encoding them), the term "identical" or "identity" percentage refers to two or more sequences or subsequences being identical or having a specified percentage of identical amino acid residues or nucleotides when compared and aligned to obtain the maximum correspondence, as measured by visual inspection or using one of the following sequence comparison algorithms.

[0030] For sequence comparisons, typically one sequence is used as a reference sequence, and the test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity between one or more test sequences and the reference sequence based on the specified program parameters.

[0031] Optimal sequence alignment can be performed for comparison, for example, using the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package of the Genetics Computer Group at 575 Science Dr., Madison, Wisconsin), or through visual inspection (see generally Current Protocols in Molecular Biology, edited by FM Ausubel et al., Current Protocols, Greene Publishing Associates, Inc. with John...). A joint venture between Wiley & Sons, Inc. (1995 Supplement) (Ausubel).

[0032] Examples of suitable algorithms for determining sequence identity percentages and sequence similarity are the BLAST and BLAST 2.0 algorithms, described by Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short codewords of length W in the query sequence that match or meet a positive threshold score T when aligned with codewords of the same length in a database sequence. T is called the adjacent codeword score threshold (Altschul et al., ibid.). These initial adjacent codeword hits are used as seeds to begin the search for longer HSPs containing them. The codeword hits are then extended in both directions along each sequence as much as possible to increase the cumulative alignment score.

[0033] For nucleotide sequences, cumulative scores are calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Word hit extension in each direction is stopped when the cumulative alignment score decreases by an amount X from its maximum attainable value; when the cumulative score becomes zero or below due to the accumulation of one or more negatively scored residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the alignment sensitivity and speed. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and two-strand comparisons as default settings. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSEIM62 scoring matrix as default settings (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. ETSA 89: 10915 (1989)).

[0034] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat. Acad. Sci. ETSA 90:5873-5787(1993)). One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in the comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0035] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid has immune cross-reactivity with the polypeptide encoded by the second nucleic acid, as described below. Therefore, for example, if the difference between one polypeptide and the second polypeptide lies only in conserved substitutions, the two peptides are generally substantially identical. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0036] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art will readily understand that nucleic acids are polynucleotides, and that polynucleotides can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant methods (i.e., using conventional cloning techniques and PCR). ™ (e.g., cloning nucleic acid sequences from recombinant libraries or cell genomes) and through synthetic means.

[0037] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers to short chains commonly referred to in the art, such as peptides, oligopeptides, and oligomers, as well as long chains of many types commonly referred to in the art as proteins. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0038] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to a target antigen, and in a particularly preferred embodiment described herein, the antigen is cMET. In this regard, the antigen-binding fragment of an antibody described herein may comprise the V-cell described herein from an antibody that binds to cMET. H and / or V L The sequence may contain one, two, three, four, five, or all six CDRs. The antigen-binding fragment of the cMET-specific antibody described herein is capable of binding to cMET. In other embodiments, the binding of the antigen-binding fragment prevents or inhibits the binding of one or more cMET ligands to the cMET receptor, thereby interrupting the biological response that would otherwise be induced by ligand-receptor binding. In some embodiments, the antigen-binding fragment specifically binds to cMET and / or inhibits or modulates the biological activity of cMET.

[0039] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binder (such as an antibody) and can also be used in animals to generate antibodies that can bind to epitopes of that antigen. An antigen may have one or more epitopes.

[0040] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to immunoglobulins or T-cell receptors. An epitope is an antigenic region that is bound by an antibody. In some embodiments, the epitope determinant comprises chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments may have specific three-dimensional structural features and / or specific charge features. In some embodiments, it is said that the antibody specifically binds to the antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. According to some embodiments, the equilibrium dissociation constant for antibody-antigen binding is less than or equal to 10. -6 M or less than or equal to 10 -7 M or less than or equal to 10 -8 When M occurs, it can be said that the antibody specifically binds to the antigen.

[0041] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer encoded information into a host cell. The term "expression vector" refers to a vector containing a nucleic acid sequence suitable for transformation into a host cell and which guides and / or controls the expression of a heterologous nucleic acid sequence. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing (if introns are present).

[0042] cMET "Human cMET" refers to the human receptor tyrosine kinase mesenchymal-epithelial transition factor. The amino acid sequence of human cMET (SEQ ID NO: 1) can also be found in GenBank: AAI30421.1.

[0043] Antibody This disclosure generally relates to isolated anti-cMET antibodies, nucleic acids encoding said antibodies and expression vectors, recombinant cells containing said vectors, and compositions comprising said antibodies. Methods for preparing said antibodies are also provided. The antibodies of this disclosure possess one or more desired functional properties, including but not limited to high affinity binding to cMET, high specificity for cMET, and the ability to inhibit HGF-cMET interaction and its downstream signaling activity.

[0044] In one general aspect, this disclosure relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to cMET.

[0045] As used herein, the term "antibody" is used broadly and includes immunoglobulins or antibody molecules, including monoclonal or polyclonal human antibodies, humanized antibodies, complex antibodies, chimeric antibodies, and antibody fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. Antibody structures are well known. Based on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five main classes (i.e., IgA, IgD, IgE, IgG, and IgM). IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibodies of this disclosure can belong to any of the five main classes or corresponding subclasses. Preferably, the antibodies of this disclosure are IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of the constant domain, the antibody light chains of vertebrate species can be classified into two distinct types (i.e., κ and λ). Therefore, the antibodies of this disclosure can contain either the κ or λ light chain constant domain. According to a specific embodiment, the antibody of this disclosure comprises a heavy chain and / or light chain constant region derived from a rat or human antibody. In addition to the heavy chain and light chain constant domains, the antibody also contains an antigen-binding region composed of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0046] Furthermore, the scope of antibodies disclosed herein also includes various forms, such as antibody fragments selected from Fv, scFv, Fab, Fab', F(ab')2, and xFab. The scope of antibodies disclosed herein also includes their derived forms, such as multispecific antibodies, or antibody derivatives further linked to other reagents, such as antibody-drug conjugates (ADCs), etc.

[0047] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to cMET is substantially free of antibodies that do not bind to cMET). Additionally, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0048] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical, except for the possibility of naturally occurring mutations present in small amounts. The monoclonal antibodies of this disclosure can be prepared using hybridoma methods, phage display technology, single-lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic nonhuman animals (such as transgenic mice or rats) having a genome containing human heavy-chain and light-chain transgenes.

[0049] As used herein, the term "antigen-binding fragment" refers to an antibody fragment such as a biantibody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv1), disulfide-stabilized biantibody (ds biantibody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent biantibody), multispecific antibody formed from an antibody moiety containing one or more CDRs, camel-derived single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody or the parent antibody fragment binds to. According to the embodiments, antigen-binding fragments include Fab and F(ab').

[0050] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region or comprises only a heavy chain variable region.

[0051] As used herein, the term "human antibody" refers to an antibody produced by a human being or an antibody having an amino acid sequence corresponding to that of a human-produced antibody prepared using any technique known in the art. This definition of human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide.

[0052] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with human antibodies, thereby retaining the antigen-binding properties of the antibody but with reduced antigenicity in the human body.

[0053] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of the light and heavy chains typically correspond to the variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of antibodies derived from another mammalian species (e.g., human) to avoid triggering an immune response in that species.

[0054] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are located on the same antigen (e.g., the same protein (or a subunit of a polyprotein)). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are located on different antigens (e.g., different proteins (or different subunits of a polyprotein)). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0055] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In one embodiment, the first and second epitopes are located on the same antigen (e.g., the same protein (or a subunit of a polyprotein)). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are located on different antigens (e.g., different proteins (or different subunits of a polyprotein)). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises a hapten or a fragment thereof having binding specificity to the first epitope and a hapten or a fragment thereof having binding specificity to the second epitope. In one embodiment, the bispecific antibody comprises an scFv or a fragment thereof that has binding specificity to a first epitope and an scFv or a fragment thereof that has binding specificity to a second epitope.

[0056] As used herein with respect to antibodies, the term "specific binding" refers to an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, this cross-species reactivity itself does not change the antibody's specific classification. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the antibody's specific classification. In some cases, the term "specific binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure, rather than a protein in general. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0057] In some embodiments, antibodies and their antigen-binding fragments, as described herein, comprise sets of heavy and light chain CDRs, respectively inserted between heavy and light chain framework regions (FRs), which support the CDRs and define their spatial relationship relative to each other. As used herein, the term "CDR set" refers to three hypervariable regions of the V region of the heavy or light chain. Starting from the N-terminus of the heavy or light chain, these regions are designated as "CDR1," "CDR2," and "CDR3," respectively. Thus, the antigen-binding site comprises six CDRs, including sets of CDRs from the respective V regions of the heavy and light chains. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analyses of many antigen-antibody complexes have demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0058] As used herein, the term "FR group" refers to the four flanking amino acid sequences of the CDR framework, which forms the V region of the heavy or light chain. Some FR residues can contact the bound antigen; however, the FRs are primarily responsible for folding the V region into the antigen-binding site, especially the FR residues directly adjacent to the CDR. Within the FR, certain amino residues and certain structural features are highly conserved. In this respect, all V region sequences contain an internal disulfide ring of approximately 90 amino acid residues. When the V region folds into the binding site, the CDR appears as a protruding ring motif forming the antigen-binding surface. It is generally accepted that there are conserved structural regions in the FR that influence the CDR ring folding shape into certain "canonical" structures, regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to participate in the non-covalent interdomain contact that stabilizes the interaction between the antibody heavy and light chains.

[0059] The structure and location of the immunoglobulin variable region can be determined by referring to the following: Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, 1987 and later (now available on the Internet (immuno.bme.nwu.edu)); Chothia, AbM, and IMGT (see, for example, Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997). ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev.Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme). Definitions of antigen-binding sites are also described in the following literature: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272. (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).For example, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (according to Kabat numbering). According to IMGT, the CDR regions of the antibody can be determined using the IMGT / DomainGap Align procedure. Unless otherwise specified, the locations of the CDRs and frame regions disclosed herein are determined according to the IMGT numbering scheme.

[0060] As used herein, “antibody heavy chain” refers to the larger of two types of polypeptide chains present in their naturally occurring conformation in all antibody molecules. Heavy chains from any vertebrate species can be classified into one of five distinct classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated as α, δ, ε, γ, and μ, respectively. Based on sequence and functional differences, the IgG and IgA classes are further subdivided into subclasses. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0061] As used in this article, "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in their naturally occurring conformation in all antibody molecules. κ and λ light chains refer to the two main isotypes of antibody light chains.

[0062] As used herein, the term "synthetic antibody" means an antibody produced using recombinant DNA technology, such as antibodies expressed by bacteriophages as described herein. The term should also be interpreted as meaning an antibody produced by synthesizing a DNA molecule encoding an antibody (and said DNA molecule expressing an antibody protein) or specifying an amino acid sequence of an antibody, wherein said DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0063] The antibody disclosed herein can be fused to another protein at its N-terminus or C-terminus (Clinical Cancer Research, 2004, 10, 1274-1281). The protein to be fused can be appropriately selected by those skilled in the art.

[0064] In a preferred embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The polypeptide sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 7, 8, 9, 12, 13, and 14, respectively, wherein the antibody or its antigen-binding fragment specifically binds to cMET, preferably human cMET, and the position of the CDR is determined according to the Kabat numbering scheme.

[0065] According to another specific aspect, this disclosure relates to isolated monoclonal antibodies or antigen-binding fragments thereof, said isolated monoclonal antibodies or antigen-binding fragments thereof comprising a heavy chain variable region having at least about 85%, preferably at least about 90%, more preferably at least about 95% or more (such as 95%, 96%, 97%, 98% or 99%) the same polypeptide sequence as SEQ ID NO: 5, or a light chain variable region having at least about 85%, preferably at least about 90%, more preferably at least about 95% or more (such as 95%, 96%, 97%, 98% or 99%) the same polypeptide sequence as SEQ ID NO: 10.

[0066] In this disclosure, the antibodies also include conserved variants thereof, which are defined as having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced with amino acids having similar or identical properties compared to the amino acid sequence of the antibodies of this disclosure to form a polypeptide. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0067] Table A

[0068] This disclosure relates to isolated nucleic acids that encode the monoclonal antibodies or antigen-binding fragments thereof disclosed herein. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., by substitution, deletion, insertion, etc.) without changing the amino acid sequence of the protein. Therefore, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibodies or antigen-binding fragments thereof disclosed herein can be altered without changing the amino acid sequence of the protein.

[0069] Polynucleotides, vectors, host cells, and preparation methods This disclosure also provides polynucleotides encoding any of the antibodies disclosed herein. In some embodiments, this disclosure discloses isolated polynucleotides encoding antibodies or antibody fragments that bind to cMET, wherein said antibody or antibody fragment comprises three light chain CDRs having the amino acid sequences shown in SEQ ID NO: 12, 13, and 14; and / or three heavy chain CDRs having the amino acid sequences shown in SEQ ID NO: 7, 8, and 9. In some embodiments, this disclosure discloses isolated polynucleotides encoding antibodies or antibody fragments that bind to cMET, wherein said antibody or antibody fragment comprises three light chain CDRs having the amino acid sequences shown in SEQ ID NO: 12, 13, and 14; and three heavy chain CDRs having the amino acid sequences shown in SEQ ID NO: 7, 8, and 9. In some embodiments, this disclosure discloses isolated polynucleotides encoding antibodies or antibody fragments that bind to cMET, wherein said antibody or antibody fragment comprises heavy chain variable regions and light chain variable regions having polypeptide sequences selected from SEQ ID NO: 5 and 10. In some embodiments, this document discloses isolated polynucleotides encoding antibodies or antibody fragments that bind to cMET, comprising SEQ ID NO: 6 and / or 11.

[0070] This disclosure also provides vectors comprising isolated nucleic acid molecules encoding monoclonal antibodies or antigen-binding fragments thereof of this disclosure. Any vector known to those skilled in the art in light of this disclosure, such as plasmids, granules, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements that establish the conventional function of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible, or repressive promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and may be used herein to generate antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis may be used to generate recombinant expression vectors according to embodiments of this disclosure. Such techniques are well known to those skilled in the art in light of this disclosure.

[0071] This disclosure also provides a host cell containing an isolated nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof of this disclosure. Those skilled in the art will recognize that any host cell known from the text of this disclosure can be used for recombinant expression of the antibodies or antigen-binding fragments thereof of this disclosure. In some embodiments, the host cell is *Escherichia coli* (E. coli). E. coliTG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 cells, 293F cells, CHO-K1 cells, or HEK293 cells (for expressing, for example, full-length IgG antibodies). According to the implementation scheme, the recombinant expression vector is transformed into host cells using conventional methods (such as chemical transfection, heat shock, or electroporation), where it is stably integrated into the host cell genome, enabling efficient expression of the recombinant nucleic acid.

[0072] This disclosure also provides a method for generating the monoclonal antibody or antigen-binding fragment thereof of the present disclosure, the method comprising culturing cells containing nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof under conditions for generating the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cells or cell culture (e.g., from a supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.

[0073] As those skilled in the art will understand, polynucleotides may include genomic sequences that express or are suitable for expressing proteins, polypeptides, peptides, etc., extragenomic and plasmid-encoded sequences, and smaller engineered gene segments. Such segments may be naturally isolated or synthesized and modified by those skilled in the art.

[0074] As those skilled in the art will recognize, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules can include hnRNA molecules containing introns and corresponding one-to-one with DNA molecules and mRNA molecules without introns. Additional coding or non-coding sequences may, but need not, be present within the polynucleotide according to this disclosure, and the polynucleotide may, but need not, be linked to other molecules and / or supporting material. Polynucleotides may contain natural sequences or sequences that may contain variants or derivatives encoding such sequences.

[0075] Typically, polynucleotide variants will contain one or more substitutions, additions, deletions, and / or insertions, preferably such that the binding affinity of the antibody encoded by the variant polynucleotide is substantially not diminished relative to the antibody encoded by the polynucleotide sequence specifically described herein.

[0076] The polynucleotides or fragments thereof described herein (regardless of the length of the coding sequence itself) can be combined with other DNA sequences (such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding regions, etc.) such that their total lengths can vary considerably. Therefore, it is anticipated that nucleic acid fragments of almost any length can be used, with the total length preferably limited by ease of preparation and use in the intended recombinant DNA scheme. For example, illustrative polynucleotide segments with total lengths of approximately 10,000, 5,000, 3,000, 2,000, 1,000, 500, 200, 100, and 50 base pairs (including all intermediate lengths) are useful.

[0077] Site-specific mutagenesis allows mutants to be generated by using a specific oligonucleotide sequence encoding the DNA sequence for the desired mutation, along with a sufficient number of adjacent nucleotides, to provide primer sequences of sufficient size and sequence complexity to form stable double strands on either side of the pierced deletion link. Mutations can be employed in selected polynucleotide sequences to improve, alter, reduce, modify, or otherwise alter the properties of the polynucleotide itself, and / or change the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0078] Antibody-dependent cell-mediated cytotoxicity (ADCC) Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated response in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize binding antibodies on target cells, subsequently causing target cell lysis. In a preferred embodiment, such cells are human cells. While not intended to be limited to any particular mechanism of action, these ADCC-mediating cytotoxic cells typically express Fc receptors (FcRs). Primary NK cells used to mediate ADCC express FcγRIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess the ADCC activity of a molecule, in vitro ADCC assays, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo (e.g., in animal models, such as those disclosed in Clynes et al., PNAS (USA), 95:652-656 (1998)).

[0079] "Effective cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRI, FcγRII, FcγRIII, and / or FcγRIV and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. In a preferred embodiment, the effector cells are human cells.

[0080] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are FcRs (γ receptors) that bind to IgG antibodies, and include receptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain. (See Daëron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are reviewed in Ravetech and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). The term “FcR” used in this paper covers other FcRs, including those to be identified in the future. The term also includes neonatal receptor FcRn responsible for transferring maternal IgG to the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

[0081] Complement-dependent cytotoxicity (CDC) Complement-dependent cytotoxicity (CDC) refers to the ability of a molecule to initiate complement activation and cleave its target in the presence of complement. The complement activation pathway is initiated by binding the first component (C1q) of the complement system to a molecule (e.g., an antibody) that has a complex with a homologous antigen. To assess complement activation, CDC assays can be performed, for example as described in Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996).

[0082] Pharmaceutical Composition This disclosure also provides pharmaceutical compositions comprising an isolated monoclonal antibody or an antigen-binding fragment thereof of the present disclosure and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising the active ingredient of the present disclosure and a pharmaceutically acceptable carrier, wherein the active ingredient is selected from: isolated monoclonal antibodies or antigen-binding fragments thereof, recombinant proteins, isolated nucleic acids (especially DNA or RNA), or carriers. The active ingredients of the present disclosure and compositions comprising them can also be used to manufacture medicaments for the therapeutic applications mentioned herein.

[0083] Therapeutic uses cMET signaling is an important pathway in the pathogenesis of cancer. In some embodiments, the antibodies or antigen-binding fragments of this disclosure can be used to prepare medicaments for treating cancer. In some embodiments, this disclosure provides the use of the antibodies or antigen-binding fragments described herein in the preparation of medicaments for treating cancer.

[0084] As used in this article, “treatment” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0085] The amount administered will depend on variables such as the type and extent of the disease or indication to be treated, the patient’s overall health status, the in vivo potency of the antibody, the drug formulation, the serum half-life of the antibody, and the route of administration.

[0086] The frequency of administration can vary depending on factors such as the route of administration, dosage, serum half-life of the antibody or fusion protein, and the disease being treated.

[0087] Example This disclosure is further described with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or those recommended by the manufacturer.

[0088] Example 1: Preparation of anti-cMET monoclonal antibody mouse immunization The RenLite® Biocytogen immunogenic mouse is a humanized mouse whose light chain variable region is genetically modified to contain only a specific combination of human VJ genes, while the heavy chain variable region contains germline genes for all human V, D, and J antibodies. The immunogen is a fusion protein (internal production, SEQ ID NOs: 2 and 3) of the extracellular segment of human cMET protein (amino acid sequence SEQ ID NO: 1, positions 1-932) and his tag or mouse Fc. The first immunogen is emulsified with an equal volume of complete Freund's adjuvant (F5881, Sigma) and injected subcutaneously at multiple sites in the abdomen. Two weeks later, an immunization dose of 25 μg is emulsified with an equal volume of incomplete Freund's adjuvant (F5506, Sigma) and injected subcutaneously at multiple sites in the abdomen.

[0089] Serum anti-cMET antibody titers were detected using ELISA to monitor humoral immune responses. Animals with high ELISA titers were selected from those meeting the titer criteria for initial immunization to screen for antibodies.

[0090] Table 1. Antigen amino acid sequence

[0091] Plasma cell screening using the Beacon single-cell light guide system Three to four days after the immunization, spleens were collected and homogenized into a single-cell suspension. Plasma cells were isolated using a mouse CD138 positive selection kit (STEMCELL™). Cells at a density of 6.25 x 10⁻⁶ cells were collected. 6Enriched plasma cells ( / ml) were introduced into the channel and placed into the NanoPen chamber of an OptoSelect 14K Chip™ (Berkeley Lights). To screen plasma cells secreting antibodies specific to human cMET protein, human cMET protein (MET-H82E1, Acrobiosystems) conjugated microbeads (520-00053, Berkeley Lights) and Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) were introduced into the channel at a protein density of 5 μg / ml. After introduction, the cryovalve was opened, and the FITC channel exposure time was set to 1000 ms. Positive signals from plasma cells were captured using 10-times-per-3-minute time-lapse imaging. After screening for human cMET protein-specific plasma cells, cynomolgus monkey cMET extracellular fragment protein (internal production, SEQ ID NO: 4) was used for further screening. Subsequently, non-specific binding signals were removed using unrelated his-tag fusion protein conjugated microbeads. Finally, plasma cells showing double positive signals for cMET protein in humans and cynomolgus monkeys were individually exported into 96-well plates filled with lysis buffer.

[0092] Example 2: Antibody heavy and light chain gene amplification, sequencing, and antibody expression First-strand cDNA was synthesized, and total cDNA was amplified using the Opto Plasma B Discovery cDNA Synthesis Kit™ (Berkeley Lights). Antibody VH and VL genes were amplified using the Opto Plasma B Discovery Sanger Prep Kit™ (Berkeley Lights). The amplified VH and VL genes were cloned into mammalian expression vectors containing the human heavy chain constant region gene IgG1 (SEQ ID NO: 15) and the Kappa chain constant region gene (SEQ ID NO: 16), respectively, and sequenced. The three heavy chain complementarity-determining regions, three light chain complementarity-determining regions, amino acid sequences of heavy chain VH and light chain VL, and DNA sequences of VH and VL are listed in Table 2 (SEQ ID No: 5-14). Antibodies were expressed in Expi293™ cells and purified by affinity chromatography.

[0093] Table 2. Amino acid sequences of anti-cMET monoclonal antibodies

[0094] Example 3. Determination of the affinity and specificity of anti-cMET antibody The affinity and specificity of the purified anti-cMET antibody were determined using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). First, 2 μg / ml of the antigen protein was coated onto 96-well ELISA plates. The antigen proteins used included human or cynomolgus monkey cMET monomeric protein cMET-ECD-his or cyno cMET-ECD-his (both internally produced, SEQ ID Nos: 2 and 4) or human cMET dimer protein formed by mouse Fc tagging. After overnight coating at 4°C, 50 μL of serially diluted antibody was added and incubated for 30–60 minutes. After washing, HRP-conjugated goat anti-human IgG secondary antibody (Abcam, ab98624) was added and incubated for 30 minutes. After washing, HRP chromogenic buffer was added for colorimetric reaction. After terminating the reaction, the absorbance was measured using a microplate reader at a wavelength of 450 nm. For antibodies that tested positive by ELISA, serial dilutions were incubated with the human gastric cancer cell line Hs746T, which highly expresses cMET, at 4°C for 30 minutes. After washing twice with FACS buffer, diluted Alexa Fluor 647-conjugated goat anti-human IgG secondary antibody (109-605-098, Jackson ImmunoResearch) was added, and the cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice more with FACS buffer, resuspended, and read using a Becton Dickinson LSR Fortessa™ cell analyzer. The non-specific binding of these antibodies was assessed by their binding to Jurkat cells (cMET negative). Nonlinear fitting was performed using GraphPad™ software to generate titration curves.

[0095] As shown in Tables 3 and 4, the antibody specifically binds to both cMET monomers and dimer proteins, exhibiting high affinity. It also specifically binds to human cMET-positive cancer cells and cross-binds with cynomolgus monkey cMET. Table 3. Binding affinity of anti-human cMET antibody to human and cynomolgus monkey cMET protein

[0096] Table 4. Binding affinity of anti-human cMET antibody to Hs746T and Jurkat cells

[0097] nd: Not detected Example 4. Binding kinetics of anti-cMET antibody The affinity and binding kinetics of purified anti-cMET antibodies were determined using surface plasmon resonance (Biacore 8K, GE Life Sciences). After capturing purified anti-cMET antibodies on a CM5 biosensor chip (Cat.No. BR100530, GE Life Sciences) immobilized with mouse anti-human Fc antibody, different concentrations of human cMET monomer His-tagged fusion protein (internal generation, SEQ ID NO: 2) were injected and incubated to assess their affinity. Changes in the surface plasmon resonance signal were analyzed using a 1:1 Langmuir binding model (BIA Evaluation Software™, GE Life Sciences), and the binding constant was calculated. k on ) and dissociation constant ( k off ).by k off / k on Ratio calculation of equilibrium dissociation constant ( K D The affinity and kinetic constants of all candidate anti-cMET monoclonal antibodies are shown in Table 5.

[0098] Table 5. Affinity and kinetic constants between anti-cMET monoclonal antibodies and cMET monomeric protein

[0099] Example 5. Anti-cMET monoclonal antibody ligand-mediated signal transduction blocking experiment To evaluate the interference of anti-cMET antibodies on HGF-cMET interaction and their effect on inhibiting downstream signaling activity, we used the inhibition of HGF-induced ERK phosphorylation at the T202 / Y204 site as a cellular readout. Specifically, H596 cells (an epithelial-like cell line isolated from human lung cancer) were seeded in 96-well plates and incubated at 37°C with 5% CO2. After 24 hours, the cells were starved in culture medium without fetal bovine serum for 4 hours. Subsequently, serially diluted anti-cMET antibody was added to each well, and the cells were incubated at 37°C for 1 hour. 100 pM of human HGF was added to each well, and the cells were incubated again at 37°C for 15 minutes. Afterward, the culture medium was discarded, 1× lysis buffer was added to the 96-well plates, and the cells were incubated at room temperature for 30 minutes. The phosphorylated ERK level in the cells was detected using the ERK phosphorylation T202 / Y204 kit (PerkinElmer).

[0100] The dose-response data were fitted to a four-parameter logistic model using GraphPad Prism software to determine the IC50 value. The formula for calculating Imax is: % Inhibition Rate = 100. [1-(X-MIN) / (MAX-MIN)], where X is the FRET signal at a given compound concentration; MAX is the signal measured in the presence of H596 cells and 100 pM human HGF; and MIN is the background signal recorded only in the presence of starved culture medium. The IC50 and Imax values ​​of the monoclonal anti-cMET antibody are detailed in Table 6.

[0101] Table 6. Comparison of anti-cMET monoclonal antibodies in terms of ligand-mediated signal transduction blocking activity

[0102] It should be understood that after reading the teachings of this disclosure, those skilled in the art can make various modifications and changes to this disclosure, and these equivalent solutions also fall within the scope defined by the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human cMET, comprising: Heavy chain variable region (VH), the heavy chain variable region comprising CDR-H1 as shown in SEQ ID NO: 7, CDR-H2 as shown in SEQ ID NO: 8, and CDR-H3 as shown in SEQ ID NO: 9; and The light chain variable region (VL) includes CDR-L1 as shown in SEQ ID NO: 12, CDR-L2 as shown in SEQ ID NO: 13, and CDR-L3 as shown in SEQ ID NO:

14.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO:

10.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising: a heavy chain constant region as shown in SEQ ID NO: 15 and a light chain constant region as shown in SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain constant region derived from IgG1, IgG2, IgG3 or IgG4 subclasses.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain constant region derived from a λ light chain or a κ light chain.

6. The antibody or its antigen-binding fragment according to claim 1, wherein it is a full-length antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody fragment is selected from Fv, scFv, Fab, Fab', and F(ab')2.

8. The antibody or its antigen-binding fragment according to claim 1 is a multispecific antibody.

9. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

10. The polynucleotide of claim 9, comprising: SEQ ID NO: 6 and / or 11.

11. A vector comprising the polynucleotide according to claim 9 or 10.

12. The vector according to claim 11, wherein it is a plasmid or a virus.

13. A host cell comprising the polynucleotide of claim 9 or 10 or the vector of claim 11 or 12.

14. The host cell according to claim 13, wherein it is a eukaryotic cell.

15. The host cell according to claim 13, wherein it is a CHO cell.

16. The host cell according to claim 13, wherein it is a prokaryotic cell.

17. The host cell according to claim 13, wherein it is Escherichia coli.

18. A method for generating an antibody or an antigen-binding fragment thereof, comprising: (a) Culture the host cell according to any one of claims 13-17 under conditions suitable for expressing the antibody or its antigen-binding fragment, and (b) optionally, recover the antibody or its antigen-binding fragment.

19. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-8, a polynucleotide according to claim 9 or 10, a vector according to claim 11 or 12, or a host cell according to any one of claims 13-18.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the polynucleotide according to claim 9 or 10, in the preparation of a medicament for treating cancer.

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