Novel immunoconjugate targeting c-met and use thereof

Abstract

The present disclosure relates to an immunoconjugate targeting c-MET, specifically an immunoconjugate comprising an anti-c-MET antibody having a specific CDR sequence comprising a terminal GlcNAc moiety, or an antigen-binding fragment thereof, a linker, and a cytotoxic drug moiety, and a pharmaceutical composition for preventing or treating cancer comprising the immunoconjugate, as an active ingredient. The immunoconjugate targeting c-MET of the present disclosure kills cancer cells at a higher level in vivo compared to other c-MET targeting ADCs and is highly stable in the body, and thus can be very useful for preventive or therapeutic use in cancer.

Description

NOVEL IMMUNOCONJUGATE TARGETING C-MET AND USE THEREOF

[0001] The present disclosure relates to an immunoconjugate targeting c-MET, specifically an immunoconjugate comprising an anti-c-MET antibody having a specific CDR sequence comprising a terminal GlcNAc moiety, or an antigen-binding fragment thereof, a linker, and a cytotoxic drug moiety, and a pharmaceutical composition for preventing or treating cancer comprising the immunoconjugate, as an active ingredient.

[0002]

[0003] An antibody-drug conjugate (ADC) is one of the fastest growing anticancer drugs in recent years. An ADC is composed of a cytotoxic drug (payload), an antibody (Ab), and a linker that connects the first two components, and is designed to broaden the therapeutic index of the drug by ensuring specific delivery to cells expressing the target antigen of the Ab (Joshua Z. Drago et al., Nature Reviews Clinical Oncology, 18(6), 327).

[0004] Meanwhile, c-MET is a protein encoded by the human MET gene, also known as hepatocyte growth factor receptor (HGFR), which is a single-pass plasma membrane protein with tyrosine kinase activity in its intracellular domain. c-MET is expressed primarily in epithelial cells and regulates cell growth and wound healing. Hepatocyte growth factor binds to c-MET as a ligand, forms a dimer, and activates c-MET. Aberrant regulation of c-MET, such as overexpression or aberrant activation, is associated with cancer development and worsens cancer prognosis. Overexpression of c-MET has been recognized in a variety of cancers, including NSCLC and colorectal cancer.

[0005] Telisotuzumab vedotin (Teliso-V), a c-MET-targeting ADC from AbbVie, showed high efficacy in NSCLC overexpressing c-MET, demonstrating the promise of c-MET-targeting ADCs (Camidge, D.R. et al., J. Clin. Oncol. 2022, 40: 9016). However, to date, there are no c-MET-targeting ADCs that have been approved by the US FDA, and there is still a high demand for the development of novel c-MET-targeting ADCs with superior anticancer efficacy.

[0006]

[0007] The present inventors developed a novel c-MET antibody, produced a novel c-MET-targeting immunoconjugate by conjugating the c-MET antibody with a drug-linker conjugate through enzymatic remodeling, and confirmed excellent anticancer effects of the produced immunoconjugate to complete the present invention.

[0008]

[0009] An object of the present disclosure is to provide an immunoconjugate comprising an anti-c-MET antibody or an antigen-binding fragment thereof, a linker and a cytotoxic drug moiety, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0010] Another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating cancer comprising the immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0011]

[0012] The immunoconjugate targeting c-MET of the present disclosure kills cancer cells at a higher level in vivo compared to other c-MET targeting ADCs and is highly stable in the body, and thus can be very useful for preventive or therapeutic use in cancer.

[0013]

[0014] FIG. 1 shows a schematic diagram of a phagemid vector used to construct an affinity-optimized antibody of huCM05.

[0015] FIG. 2 shows a schematic diagram of the enzymatic remodeling technique used to produce an immunoconjugate of the present disclosure.

[0016] FIG. 3 shows a process of producing the CM05-Auri immunoconjugate of the present disclosure.

[0017] FIG. 4 shows the MS analysis results identifying azido-huCM05.

[0018] FIG. 5 shows the MS analysis results identifying CM05-Auri.

[0019] FIG. 6 shows a structure of CM05-Auri.

[0020] FIG. 7 shows the results of target protein binding affinity evaluation using ELISA.

[0021] FIG. 8 shows the results of binding affinity evaluation to target protein-expressing cells using FACS.

[0022] FIG. 9 shows the results of comparative evaluation of the internalization rates of huCM05 antibody and CM05-Auri.

[0023] FIG. 10 shows the results of a serial internalization assay performed on huCM05 antibody and CM05-Auri.

[0024] FIG. 11 shows the results of confirming the bystander effect of CM05-Auri.

[0025] FIG. 12 shows the in vivo activity of CM05-Auri in the H441 CDX model.

[0026] FIG. 13 shows the in vivo activity of CM05-Auri in the H1975 CDX model.

[0027] FIG. 14 shows the in vivo activity of CM05-Auri in the HT-29 CDX model.

[0028] FIG. 15 shows the in vivo activity of CM05-Auri in the HCC827 CDX model.

[0029] FIG. 16 shows the in vivo activity of CM05-Auri in the H1573 CDX model.

[0030] FIG. 17 shows the in vivo activity of CM05-Auri in the LU11681 CDX model.

[0031] FIG. 18 shows the in vivo activity of CM05-Auri in the LU5165 CDX model.

[0032] FIG. 19 shows the results of confirming the stability of CM05-Auri in body circulation in cynomolgus monkeys.

[0033]

[0034] The present disclosure will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present disclosure may also be applied to each of the other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, it cannot be considered that the scope of the present disclosure is limited by specific descriptions described below.

[0035]

[0036] I. Immunoconjugate

[0037] One aspect of the present disclosure for achieving the above object is an immunoconjugate represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0038] [Formula 1]

[0039]

[0040] in Formula 1 above,

[0041] Ab is an antibody comprising a light chain variable region comprising light chain CDR1 set forth in SEQ ID NO: 1; light chain CDR2 set forth in SEQ ID NO: 2; and light chain CDR3 set forth in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 set forth in SEQ ID NO: 4; heavy chain CDR2 set forth in SEQ ID NO: 5; and heavy chain CDR3 set forth in SEQ ID NO: 6, an affinity-optimized antibody thereof, or an antigen-binding fragment thereof;

[0042] the Ab comprises a terminal GlcNAc moiety of the following Formula 2,

[0043] [Formula 2]

[0044]

[0045] wherein S is GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) as a sugar moiety,

[0046] Fuc is fucose, and

[0047] a is 0 or 1;

[0048] a terminal azide group of the terminal GlcNAc moiety in the Ab binds to the BCN (Bicyclo[6.1.0]non-4-yne) ring to form a triazole moiety of the following Formula 3,

[0049] [Formula 3]

[0050]

[0051] wherein * is linked to the S in the antibody and ** is linked to the SU; and

[0052] SUis a group connecting the BCN ring and LUand is represented by the following Formula 4,

[0053] [Formula 4]

[0054]

[0055] wherein * is linked to the BCN ring, and ** is linked to the LU,

[0056] RAand RBare each independently -H or -C1-3alkyl,

[0057] n is an integer from 1 to 5;

[0058] LUis a single bond or a branched linker, and

[0059] when LUis a branched linker, LUis represented by the following Formula 5,

[0060] [Formula 5]

[0061]

[0062] wherein * is linked to the SU, and ** is linked to the CL,

[0063] M1and M2are each independently -C(=O)- or -OC(=O)-,

[0064] q1and q2are each independently integers from 2 to 4; and

[0065] CLis a cleavable linker represented by the following Formula 6,

[0066] [Formula 6]

[0067]

[0068] wherein Dipeptide is -Val-Cit-, -Val-Ala-, -Phe-Lys- or -Glu-Ala-,

[0069] Rx is each independently selected from -C1-3alkyl, -halo, or -OH,

[0070] r is an integer from 0 to 4;

[0071] D is a cytotoxic drug moiety; and

[0072] p is an integer from 1 to 3.

[0073] As used herein, the term "immunoconjugate" refers to a complex in which a drug-linker conjugate is linked to an antibody or an antigen-binding fragment thereof, and specifically has the structure of Formula 1 below:

[0074] [Formula 1]

[0075] .

[0076] In the present disclosure, the term "drug-linker conjugate" refers to a material for the production of an immunoconjugate in which an antibody or an antigen-binding fragment thereof is not linked, and may be used as an immunoconjugate by binding with any antibody or antigen-binding fragment thereof depending on the purpose.

[0077] When administered in vivo, the immunoconjugate may release the drug after the antibody or the antigen-binding fragment binds to the target antigen, thereby enabling the drug to act on target cells and / or surrounding cells, resulting in excellent efficacy and reduced side effects as a targeted drug.

[0078] As used herein, the term "antibody" refers to a protein molecule that acts as a ligand to specifically recognize an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen, and includes polyclonal antibody, monoclonal antibody, and whole antibody. Further, the term includes chimeric antibody and bivalent or bispecific molecule, diabody, triabody, and tetrabody. The term further includes single-chain antibody with binding function to FcRn, scab, derivative of antibody constant region, and artificial antibody based on protein scaffold. A whole antibody is a structure having two full-length light chains and two full-length heavy chains, wherein each light chain is linked to the heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0079] As used herein, the terms "fragment", "antibody fragment" and "antigen-binding fragment" are used interchangeably to refer to any fragment of an antibody of the present disclosure that retains the antigen-binding function of the antibody. Exemplary antigen binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, dsFv, and scFv.

[0080] The Fab has one antigen binding site, which is a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region of the heavy chain (CH1 domain). An antigen-binding fragment or an antibody fragment of an antibody molecule refers a fragment that possesses antigen-binding functionality, and Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2antibody is produced when cysteine residues in the hinge region of Fab' form a disulfide bond. Fd refers to the heavy chain portion contained in the Fab fragment. Fv (variable fragment) refers to the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region. Double disulfide Fv (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond, while single chain Fv (scFv) has a heavy chain variable region and a light chain variable region linked by a covalent bond, usually through a peptide linker, or directly at the C-terminus, to form a dimer-like structure. Although not limited thereto, these antibody fragments may be obtained using proteolytic enzymes (e.g., Fab may be obtained by restriction digestion of the whole antibody with papain, and F(ab')2fragment may be obtained by digestion with pepsin), or may be produced through genetic recombination technology.

[0081] Hepatocyte growth factor receptor (c-MET) is a type of RTK and is a cell surface receptor for HGF / SF (hepatocyte growth factor known as scatter factor) (Laird AD et al., Expert. Opin. Investig. Drugs 12: 51 -64 (2003)). Aberrant activation of c-MET by HGF is one of the representative mechanisms of tumorigenesis and is known to be associated with tumor proliferation, inhibition of apoptosis, neovascularization, invasion, and metastasis (Bottaro DP et al., Science 251: 802-804 (1991), Day RM et al., Oncogene 18: 3399-3406 (1999)). In addition, aberrant activation of c-MET by mutation and amplification of c-MET is associated with a variety of cancers, including lung, colorectal, head and neck, gastric, and breast cancers, and has been reported to be associated with increased tumor aggressiveness and poor prognosis (Lefebvre J et al., FASEB J 26: 1387-1399 (2012), Liu X et al., Trends Mol Med 16: 37-45 (2010), Smolen GA et al., Proc Natl Acad Sci USA 103: 2316-2321 (2006), Foveau B et al., Mol Biol Cell 20: 2495-2507 (2009)). Therefore, c-MET is attracting attention as a target antigen for treating various cancers, and multiple approaches are being attempted to inhibit the expression and activity of c-MET. However, as described above, c-MET is associated with the development and progression of a variety of cancers, which leads to an ongoing need to develop novel therapeutic agents capable of treating cancer by targeting c-MET.

[0082] Specifically, in the present disclosure, the anti-c-MET antibody comprises a light chain variable region comprising light chain CDR1 set forth in SEQ ID NO: 1; light chain CDR2 set forth in SEQ ID NO: 2; and light chain CDR3 set forth in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 set forth in SEQ ID NO: 4; heavy chain CDR2 set forth in SEQ ID NO: 5; and heavy chain CDR3 set forth in SEQ ID NO: 6.

[0083] As used herein, the term "a heavy chain" may include full length heavy chain comprising: a variable region domain VH, which comprises amino acid sequences having enough variable region sequences to allow the specificity to an antigen, and 3 constant region domains CH1, CH2 and CH3, and fragments thereof. In addition, the term "a light chain" may include a full length light chain comprising: a variable region domain VL, which comprises amino acid sequences having enough variable region sequences to allow the specificity to an antigen, and a constant region domain CL, and fragments thereof.

[0084] In the present disclosure, the antibody may include both mouse antibodies produced from mice, and mutants obtained by substituting, adding, and / or deleting part of the amino acid sequence of the parent antibody in order to improve the affinity, immunity, etc., of the antibody. The mutants may include, but are not limited to, chimeric antibody, humanized antibody, affinity-optimized antibody, and the like.

[0085] In the present disclosure, the mutant refers broadly to an antibody which comprises the same CDR as the parent antibody, or in which a portion of the parent antibody CDR amino acid sequence is modified (substituted, added or deleted) while the target epitope remains the same. The mutant may be suitably modified by those skilled in the art to improve the affinity, immunogenicity, and the like, of the antibody within the range where the binding ability to the same epitope is maintained.

[0086] In other words, the antibody or the antigen-binding fragment of the present disclosure may comprise not only the sequences of the anti-c-MET antibodies described herein, but also biological equivalents thereof, to the extent that they are capable of specifically recognizing c-MET. For example, additional modifications may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. These amino acid modifications are made based on the relative similarity of the amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. According to analysis of the size, shape and type of amino acid side chain substitutions, it may be seen that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan, and tyrosine may be biologically functional equivalents.

[0087] As used herein, the term "chimeric antibody" refers to an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, resulting in a significantly improved immune response compared to a mouse antibody.

[0088] As used herein, the term "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species is modified to be similar to that of antibody naturally produced from human. For example, the humanized antibody may be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region and recombining the variable region with a constant region of a desirable human antibody. However, a CDR grafting alone results in a low affinity of the humanized antibody, so several important FR amino acid residues, which are believed to affect the three-dimensional structure of CDR, may be affinity-engineered to those of the mouse antibody, thereby achieving the same level as the affinity of the original mouse antibody.

[0089] As used herein, the term "affinity-optimized antibody" refers to a mutant in which a portion of the CDR sequence of a specific antibody is substituted, added, or deleted, indicating an antibody with a better binding affinity to an antigen while binding to the same antigen epitope as that of the specific antibody. Specifically, the affinity-optimized antibody in the present disclosure refers to a mutant antibody that binds to the same epitope as the antibody comprising a light chain variable region comprising light chain CDR1 set forth in SEQ ID NO: 1; light chain CDR2 set forth in SEQ ID NO: 2; and light chain CDR3 set forth in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 set forth in SEQ ID NO: 4; heavy chain CDR2 set forth in SEQ ID NO: 5; and heavy chain CDR3 set forth in SEQ ID NO: 6. A person skilled in the art can produce the affinity-optimized antibodies using known techniques based on the specified light and heavy chain CDR sequences. For example, the affinity-optimized antibodies of the present disclosure may be produced via phage display. As used in the present disclosure, the term "phage display" refers to a technique for displaying a mutant polypeptide as a fusion protein with at least a portion of an envelope protein on the surface of a phage, for example, a filamentous phage particle. The utility of phage display resides in the fact that it is possible to quickly and efficiently classify sequences that exhibit high-affinity binding to target antigens by targeting large libraries of randomized protein mutants. Displaying peptide and protein libraries on phages has been used to screen millions of polypeptides to identify those with specific binding properties.

[0090] In an embodiment of the present disclosure, the antibody may be an antibody comprising a light chain variable region set forth at SEQ ID NO. 7 and a heavy chain variable region set forth at SEQ ID NO. 8. For example, the antibody may be an antibody comprising a light chain variable region encoded by the nucleotide set forth in SEQ ID NO. 9 and a heavy chain variable region encoded by the nucleotide set forth in SEQ ID NO. 10, but is not limited thereto.

[0091] In another embodiment of the present disclosure, the antibody may comprise (a) a light chain variable region set forth in SEQ ID NO: 11 and a heavy chain variable region set forth in SEQ ID NO: 12; or (b) a light chain variable region set forth in SEQ ID NO: 15 and a heavy chain variable region set forth in SEQ ID NO: 16. For example, the antibody may comprise (a) a light chain variable region encoded by the nucleotide set forth in SEQ ID NO: 13 and a heavy chain variable region encoded by the nucleotide set forth in SEQ ID NO: 14; or (b) a light chain variable region encoded by the nucleotide set forth in SEQ ID NO: 17 and a heavy chain variable region encoded by the nucleotide set forth in SEQ ID NO: 18, but is not limited thereto. In addition, the antibody may comprise a hinge region set forth in any one of SEQ ID NO: 19 to SEQ ID NO: 26.

[0092] As another embodiment of the present disclosure, the affinity-optimized antibody for the antibody above may comprise at least one amino acid sequence substitution in an antibody comprising a light chain variable region comprising light chain CDR1 set forth in SEQ ID NO: 1; light chain CDR2 set forth in SEQ ID NO: 2; and light chain CDR3 set forth in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 set forth in SEQ ID NO: 4, heavy chain CDR2 set forth in SEQ ID NO: 5; and heavy chain CDR3 set forth in SEQ ID NO: 6, wherein (i) in the light chain CDR1, G at position 1 is substituted with A, E, K, L, N, R, S, V or W; A at position 2 is substituted with C, G, I, P, S, T or V; S at position 3 is substituted with G, M, N, P, Q, R, S or T; E at position 4 is substituted with A, D, F, G, H, K, M, Q, R, S, T or V; N at position 5 is substituted with A, D, E, G, K, L, P, Q, R, S, T or V; I at position 6 is substituted with A, F, L, M, Q, R, S, T or V; Y at position 7 is substituted with F, H, R or V; or G at position 8 is substituted with D, F, H, M, N, R, S, T or V; (ii) in the light chain CDR2, G at position 1 is substituted with D, F, H, K, P, Q, S, V or Y; T at position 3 is substituted with Q; or N at position 4 is substituted with G; (iii) in the light chain CDR3, Q at position 1 is substituted with E, G, I, M or N; N at position 2 is substituted with A, D, E, H, L, Q, S or T; V at position 3 is substituted with I, L, M, N, Q, S or T; L at position 4 is substituted with F, H, I, M, R, S, V, W or Y; S at position 5 is substituted with C, D, E, F, G, H, K, L, N, Q, R, T, V or Y; S at position 6 is substituted with D, E, F, G, H, I, L, M, N, P, Q, R, T, V or Y; P at position 7 is substituted with A, D, E, G, N, Q, S or V; Y at position 8 is substituted with E, F, L, M or Q; or T at position 9 is substituted with D, F, G, I, L, N, S, V, W or Y; (iv) in the heavy chain CDR1, D at position 1 is substituted with G or Q; Y at position 2 is substituted with Q; or I at position 4 is substituted with A or Q; (v) in the heavy chain CDR2, F at position 3 is substituted with D, E, W or Y; G at position 5 is substituted with D, H or Y; S at position 6 is substituted with F, P, W or Y; G at position 7 is substituted with A, F, L, N or T; N at position 8 is substituted with F, P, S, T or Y; T at position 9 is substituted with A, D, E, F, G, H, L, P, S or V; H at position 10 is A, D, F, M, R, S, T, V, W or Y; F at position 11 is substituted with G, H, I, L, M, N, P, Q, V or Y; S at position 12 is substituted with A, D, G, H, I, L, P, T or V; A at position 13 is substituted with D, E, F, G, H, I, K, L, M, P, R, S, T, V or Y; R at position 14 is substituted with A, E, G, H, L, N, P, Q, S, W or Y; F at position 15 is substituted with D, E, G, L, M, P, R, S, V or W; K at position 16 is substituted with A, E, F, G, H, L, R, S, T, V or Y; or G at position 17 is substituted with E, F, H, L, M, N, P, Q, R, S, T, V or W; or (vi) in the heavy chain CDR3, G at position 1 is substituted with E, F, H, N, Q, V or W; D at position 2 is substituted with E; Y at position 3 is substituted with L, Q, T or V; G at position 4 is W; F at position 5 is substituted with L or Y; L at position 6 is substituted with Q, S or Y; or Y at position 7 is substituted with C, L, M, N or Q, wherein the light chain CDR1 may comprise 0 to 5 substitutions, the light chain CDR2 may comprise 0 to 1 substitution, the light chain CDR3 may comprise 0 to 7 substitutions, the heavy chain CDR1 may comprise 0 to 1 substitution, the heavy chain CDR2 may comprise 0 to 11 substitutions, and the heavy chain CDR3 may comprise 0 to 6 substitutions, but the present disclosure is not limited thereto.

[0093] As another embodiment of the present disclosure, the affinity-optimized antibody may specifically comprise a light chain variable region comprising light chain CDR1 set forth in any one of SEQ ID NO: 1, and SEQ ID NO: 124 to SEQ ID NO: 163; light chain CDR2 set forth in any one of SEQ ID NO: 2, and SEQ ID NO: 164 to SEQ ID NO: 174; and light chain CDR3 set forth in any one of SEQ ID NO: 3, and SEQ ID NO: 175 to SEQ ID NO: 284, and a heavy chain variable region comprising heavy chain CDR1 set forth in any one of SEQ ID NO: 4, and SEQ ID NO: 36 to SEQ ID NO: 40; heavy chain CDR2 set forth in any one of SEQ ID NO: 5, and SEQ ID NO: 41 to SEQ ID NO: 110; and heavy chain CDR3 set forth in any one of SEQ ID NO: 6, and SEQ ID NO: 111 to SEQ ID NO: 123. More specifically, the affinity-optimized antibody may comprise a light chain variable region set forth in any one of SEQ ID NO: 11, and SEQ ID NO: 289 to SEQ ID NO: 294, and a heavy chain variable region set forth in any one of SEQ ID NO: 12, and SEQ ID NO: 285 to SEQ ID NO: 288. Even more specifically, the affinity-optimized antibody may comprise (a) a light chain variable region set forth in SEQ ID NO: 11 and a heavy chain variable region set forth in SEQ ID NO: 285; (b) a light chain variable region set forth in SEQ ID NO: 11 and a heavy chain variable region set forth in SEQ ID NO: 288; (c) a light chain variable region set forth in SEQ ID NO: 293 and a heavy chain variable region set forth in SEQ ID NO: 12; (d) a light chain variable region set forth in SEQ ID NO: 291 and a heavy chain variable region set forth in SEQ ID NO: 288; (e) a light chain variable region set forth in SEQ ID NO: 289 and a heavy chain variable region set forth in SEQ ID NO: 285; (f) a light chain variable region set forth in SEQ ID NO: 290 and a heavy chain variable region set forth in SEQ ID NO: 286; (g) a light chain variable region set forth in SEQ ID NO: 291 and a heavy chain variable region set forth in SEQ ID NO: 287; (h) a light chain variable region set forth in SEQ ID NO: 292 and a heavy chain variable region set forth in SEQ ID NO: 287; (i) a light chain variable region set forth in SEQ ID NO: 294 and a heavy chain variable region set forth in SEQ ID NO: 287; or (j) a light chain variable region set forth in SEQ ID NO: 289 and a heavy chain variable region set forth in SEQ ID NO: 285, but is not limited thereto.

[0094] The antibody of the present disclosure is characterized, in particular, by being modified to comprise a terminal GlcNAc (N-acetylglucosamine) moiety of the following Formula 2:

[0095] [Formula 2]

[0096] .

[0097] By modifying the antibody to have the terminal GlcNAc moiety at a specific position, the antibody may be easily combined with a desired number of drug-linker conjugates, resulting in stable preparation of immunoconjugate.

[0098] The modified antibody may comprise an asparagine residue at region 290-305 (e.g., N297; EU numbering), where a terminal GlcNAc moiety may be present. The terminal GlcNAc moiety may be linked to the antibody via the C1 position of GlcNAc.

[0099] In the present disclosure, S is a sugar moiety and includes both sugar or sugar derivative. The sugar or sugar derivative may be, but are not limited to, galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc), or sialic acid (N-acetylneuraminic acid), and all of which are included in the scope of the present disclosure, at least to an equal extent, as long as they can accomplish the same purpose as the present disclosure. In the present disclosure, the modification of the antibody may be achieved by 1) trimming a glycan structure of the antibody with endoglycosidase, and then 2) attaching a terminal GlcNAc moiety to the antibody by treating the nucleoside mono- or diphosphate-S-N3with glycosyltransferase (enzymatic remodeling). In this way, an azide group can be introduced into the antibody (azido-Ab), and the modified antibody can be simply combined with the drug-linker conjugate via a non-metallic click reaction, to facilitate the preparation of immunoconjugate (FIG. 2). The types of endoglycosidase and glycosyltransferase used to modify the antibody of the present disclosure are not limited, and a person skilled in the art may select and use an appropriate enzyme for modification of the antibody.

[0100] In Formula 2 above, a may be 0 or 1, that is, fucose (Fuc) may be absent or one fucose may be added to GlcNAc.

[0101] The nucleoside mono- or diphosphate used for antibody modification in the present disclosure may be, for example, but not limited to, uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP), or cytidine monophosphate (CMP).

[0102] In the present disclosure, the sugar derivative S-N3may be, for example, GalNAz (2-azidoacetamidogalactose), 6-AzGal (6-azido-6-deoxygalactose), 6-AzGaiNAe (6-azido-6-deoxy-2-acetamidogalactose), 4-AzGalNAc (4-azido-4-deoxy-2- acetamidogalactose), 6-AzGalNAz (6-azido-6-deoxy-2-azidoacetamidogalactose), GlcNAz (2-azidoacetamidoglucose), 6-AzGlc (6~azido~6~deoxyglucose), 6-azido-6-deoxy-2-acetamidoglucose (6-AzGlcNAc), 4-AzGlcNAc (4-azido-4-deoxy-2-acetamidoglucose) or 6-AzGlcNAz (6-azido-6-deoxy-2-azidoacetamidoglucose), but is not limited thereto. It is also to be understood that substitution of a reactive functional group, such as a keto or an alkynyl group, rather than an azide group, is also within the scope of the present disclosure as long as it has an equivalent effect.

[0103] Since the above enzymatic remodeling may theoretically occur at only two positions in one monoclonal antibody, in principle, it can be seen that there are two bindings between the antibody and BCN in the present disclosure, thereby facilitating the preparation of immunoconjugate with desired DAR value. However, it should be noted that when Ab is used in the form of an antigen-binding fragment rather than a full-length antibody, or in the form of a multivalent antibody such as a bispecific antibody, the number of bindings may vary depending on the structure and conformation of the antigen-binding fragment or multivalent antibody. In other words, the description of Formula 1 above in which Ab binds to two BCN-SU-LU-(CL-D)p is based on a monoclonal antibody and may be interpreted differently depending on the structure and conformation of Ab.

[0104] The immunoconjugate of the present disclosure can form a triazole moiety by combining the terminal azide group of the terminal GlcNAc moiety of the Ab with the BCN (Bicyclo[6.1.0]nonyne) ring or a derivative thereof, and thus it is possible to prepare the immunoconjugate through a simple non-metallic click reaction with the drug-linker conjugate. In the present disclosure, the BCN ring or the derivative thereof may be those represented by the following Formulas 7-1 to 7-3:

[0105] [Formula 7-1]

[0106]

[0107] [Formula 7-2]

[0108]

[0109] [Formula 7-3]

[0110]

[0111] in the Formulas 7-1 to Formula 7-3 above,

[0112] X is -CRD- or -N-,

[0113] RCis each independently -H, substituted or unsubstituted -C1-6alkyl, -C3-10cycloalkyl, -3 to 10-membered heterocycloalkyl, -C6-12aryl or -5 to 12-membered heteroaryl,

[0114] RDis -H, -halo or -C1-3alkyl,

[0115] Ry is each independently -halo, -OH, -OC1-3alkyl, -NO2, -CN, -S(O)2C1-3alkyl, -C1-6alkyl, -C3-10cycloalkyl, -3 to 10-membered heterocycloalkyl, -C6-12aryl or -5 to 12-membered heteroaryl, and

[0116] m is an integer from 1 to 4.

[0117]

[0118] For example, the BCN ring may be a bicyclo[6.1.0]non-4-yne, in which case it forms the triazole moiety of the following Formula 3:

[0119] [Formula 3]

[0120]

[0121] wherein * is linked to the S in the antibody and ** is linked to the SU.

[0122] In the immunoconjugate of the present disclosure, SU(Stretcher Unit) is a group connecting the BCN ring and LU, and those skilled in the art may use linker units known in the art to appropriately set the distance between the Ab and cytotoxic drug moiety in the immunoconjugate of the present disclosure. SUmay include functional group such as, for example, -CH2-, -OC(=O)-, -C(=O)O-, -NRE-, -O-, -S-, -CH2CH2O-, -OCH2CH2-, -NRF(C=O)-, -C(=O)NRG-, -C(=O)-, -S(=O)-, -S(=O)2- (wherein RE, RF, and RGare each independently -H or C1-3alkyl), but all are included within the scope of the present disclosure as long as they can achieve the same purpose as the immunoconjugate of the present disclosure.

[0123] For example, in the immunoconjugate of the present disclosure, SUmay be represented by the following Formula 4:

[0124] [Formula 4]

[0125]

[0126] wherein * is linked to the BCN ring, and ** is linked to the LU,

[0127] RAand RBare each independently -H or -C1-3alkyl, and

[0128] n is an integer from 1 to 5.

[0129]

[0130] In the immunoconjugate of the present disclosure, the linking unit (LU) is a moiety that links the SUand CL, and may be a single bond or a branched linker, depending on the purpose. In other words, those skilled in the art may adjust the drug-antibody ratio (DAR) of the immunoconjugate of the present disclosure depending on the form of the LU, wherein p is an integer from 1 to 3.

[0131] For example, but not limited thereto, when two drugs are linked via LU, the LUmay be represented by the following Formula 5, and when three drugs are linked via LU, the LUmay be represented by the following Formula 8:

[0132] [Formula 5]

[0133]

[0134] [Formula 8]

[0135]

[0136] wherein * is linked to the SU, and ** is linked to the CL,

[0137] RHis -H or -C1-3alkyl,

[0138] M1to M3are each independently -C(=O)- or -OC(=O)-, and

[0139] q1to q3are each independently integers from 2 to 4.

[0140] In the immunoconjugate of the present disclosure, CLis a cleavable linker and may include an enzymatic or non-enzymatically cleavable linker. For example, CLmay include a peptide moiety, which is a unit consisting of one or more natural or non-natural amino acids that contributes to the cleavage of the immunoconjugate of the present disclosure in the body to release the drug. For example, when the peptide moiety is a dipeptide, it may be -Val-Cit-, -Val-Ala-, -Phe-Lys-, -Glu-Ala-, -Cit-Val-, -Ala-Ala-, -Ala-Cit-, -Cit-Ala-, -Asn-Cit-, -Cit-Asn-, -Cit-Cit-, -Val-Glu-, -Glu-Val-, -Ser-Cit-, -Cit-Ser-, -Lys-Cit-, -Cit-Lys-, -Asp-Cit-, -Cit-Asp or -Ala-Val-, but is not limited thereto. The CLmay further comprise a spacer unit such as a substituted or unsubstituted p-aminobezyloxy-carbonyl (PABC) group. Further, the PABC group may be substituted with sugar or derivative thereof to form glucuronide units (see WO2007 / 011968).

[0141] As a specific example, the CLmay be a cleavable linker represented by the following Formula 6:

[0142] [Formula 6]

[0143]

[0144] wherein Dipeptide is -Val-Cit-, -Val-Ala-, -Phe-Lys- or -Glu-Ala-,

[0145] Rx is each independently selected from -C1-3alkyl, -halo, or -OH, and

[0146] r is an integer from 0 to 4.

[0147] In the immunoconjugate of the present disclosure, D refers to a cytotoxic drug moiety, which is capable of achieving the desired anticancer effect by being released within the cell, after the immunoconjugate of the present disclosure being introduced into cancer cell, or around the cancer cell. The cytotoxic drug moiety may be, for example, but not limited to, i) a DNA binding drug, ii) a kinase inhibitor, iii) a MEK inhibitor, iv) a KSP inhibitor, v) a topoisomerase inhibitor, vi) a DNA alkylating agent, vii) a PARP inhibitor, viii) a NAMPT inhibitor, ix) a protein synthesis inhibitor, or x) an immunomodulatory compound.

[0148] In an embodiment, the cytotoxic drug moiety in an immunoconjugate of the present disclosure may be an auristatin compound, a calicheamicin compound, or an anthracycline compound, and these compounds, when produced as an immunoconjugate with an antibody of the present disclosure or an antigen-binding fragment thereof, exhibits significantly superior anticancer activity than other known c-MET targeting ADCs. The auristatin compound in the present disclosure refers to an auristatin and derivative compounds thereof that are cytotoxic as tubulin inhibitors, and may be, for example, but not limited to, auristatin, dolastatin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F, AF-HPA, MMAF-HPA, or phenylenediamine (AFP). The calicheamicin compound in the present disclosure refers to a calicheamicin derived fromMicromonospora echinosporaand derivative compounds thereof that bind to the DNA minor groove and exhibit cytotoxicity. In the present disclosure, the anthracycline compound refers to a tetracyclic compound derived fromStreptomyces peucetiusand derivative compounds thereof that are inserted between DNA base pairs and exhibit cytotoxicity.

[0149] In the present disclosure, "stereoisomer" refers to a compound that has the same chemical or molecular formula but is sterically different. The stereoisomer in the present specification include optical isomer, enantiomer, diasteromer, cis / trans isomer, rotamer, and atropisomer, and each of the isomer, racemate, and mixture thereof are also included within the scope of the present disclosure. Unless otherwise specified, a solid bond ( ) connected to an asymmetric carbon atom may include a wedge solid bond or wedge dashed bond representing the absolute arrangement of stereocenter.

[0150] In the present disclosure, "pharmaceutically acceptable salt" refers to salts commonly used in the pharmaceutical industry, and may include, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, and the like, and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid, and in addition thereto, may include salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotate acid, acetylsalicylic acid, and amino acid salts such as lysine, arginine, guanidine, and the like. Further, organic ion salts such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyl trimethyl ammonium, and benzethonium that can be used in pharmaceutical reactions, purification, and separation processes, may be included. However, the types of salts meant in the present disclosure are not limited by these listed salts.

[0151]

[0152] II. Pharmaceutical composition

[0153] Another aspect of the present disclosure for achieving the above object is a pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0154] Another aspect of the present disclosure is a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof.

[0155] Another aspect of the present disclosure is the use of the immunoconjugate to prevent or treat cancer. Still another aspect of the present disclosure is the use of the immunoconjugate for preparing the pharmaceutical composition.

[0156] The immunoconjugate, stereoisomers thereof, and pharmaceutically acceptable salts thereof are the same as described above.

[0157] The immunoconjugate of the present disclosure specifically binds to c-MET and induces cancer cell death, and thus may be usefully employed in the treatment or prevention of cancer.

[0158] In the present disclosure, the cancer may be solid cancer or hematological cancer. For example, the cancer may be one or more types selected from the group consisting of pseudomyxoma, intrahepatic bile duct cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell cancer, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, mastocytoma, sinonasal cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, childhood leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid, gastrointestinal stromal tumor, gastrointestinal stromal cancer, Wilms cancer, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, acoustic nerve schwannoma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsillar cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleura cancer, blood cancer, and thymic cancer, but is not limited thereto.

[0159] The cancer may be a c-MET positive cancer and may include all cancers related to the c-MET gene.

[0160] As used herein, the term "prevention" means any act of inhibiting or delaying the progression of a cancerous disease by administration of the composition according to the present disclosure, and the term "treatment" means inhibiting the development of a cancerous disease, alleviating or eliminating a cancerous disease.

[0161] As used herein, the term "therapeutically effective amount" refers to an amount of the immunoconjugate that is effective for the treatment or prevention of cancer. Specifically, the "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined depending on factors including the subject type and severity, age, sex, type of diseases, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and when combined, may be administered sequentially or simultaneously. In addition, the pharmaceutical composition of the present disclosure may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the pharmaceutical composition in an amount capable of achieving maximum effect with the minimum amount without side effects, and the dose may be easily determined by those skilled in the art depending on various factors such as the patient's condition, age, gender, and complications.

[0162]

[0163] Exemplary embodiments of the present disclosure may be modified in various other forms, and the scope of the present disclosure is not limited to the exemplary embodiments described below. In addition, the exemplary embodiments of the present disclosure are provided to more completely explain the present disclosure to an ordinary person skilled in the art. Further, "comprising" a component throughout the specification does not mean excluding other components, but rather means that other components may be further included, unless otherwise stated.

[0164]

[0165] Hereinafter, the constitution and effects of the present disclosure will be described in more detail through the following Examples. These Examples are only provided for illustrating the present disclosure, but the scope of the present disclosure is not limited by these Examples.

[0166]

[0167] Example 1. Preparation of hybridoma cells producing c-MET specific antibodies and confirmation of tumor cell proliferation inhibitory activity thereof

[0168] (1) Preparation and screening of hybridoma cell line producing monoclonal antibodies against c-MET protein

[0169] To obtain immunized mice for the development of hybridoma cell line through animal immunization, mice were intraperitoneally injected with human c-MET Sema domain / Fc fusion protein (made in-house) as an antigen. In order to select hybridoma cells that specifically respond only to the c-MET protein among the hybridoma cell population, screening was performed by ELISA assay using human c-MET / His fusion protein as antigen.

[0170]

[0171] (2) c-MET antibody

[0172] The light-chain and heavy-chain CDR amino acid sequences of mouse antibody mCM05 obtained from the selected hybridoma cell line are shown in Table 1.

[0173]

[0174] CDR1CDR2CDR3Light chainGASENIYGALN(SEQ ID NO: 1)GATNLAD(SEQ ID NO: 2)QNVLSSPYT(SEQ ID NO: 3)Heavy chainDYYIN(SEQ ID NO: 4)EIFPGSGNTHFSARFKG(SEQ ID NO: 5)GDYGFLY(SEQ ID NO: 6)

[0175]

[0176] (3) In vitro tumor cell proliferation inhibitory activity of hybridoma c-MET antibody

[0177] The tumor cell proliferation inhibitory activity of the c-MET-specific mouse antibody (mCM05) obtained using the hybridoma cell line and the chimeric antibodies (cCM05-IgG1, cCM05-IgG2) produced by fusing the antibody with human heavy and light chain constant regions was tested in U-87 MG, a human glioblastoma cell line, and MKN45, a human gastric cancer cell line.

[0178] Specifically, U-87 MG cells (ATCC, #HTB14) diluted in culture medium EMEM (ATCC, #30-2003) containing 10% (v / v) FBS, 100 U / 400 ml penicillin and 100 μg / 500 ml streptomycin (Invitrogen, #15140-122) were added in 100 μl at a concentration of 2.5 Х 103cells per well of a 96-well plate, and the plate was incubated at 37℃, 95% relative humidity and 5% (v / v) CO2for 18-24 hours. After removing the cell culture medium from each well, 100 μl of EMEM medium containing 2% (v / v) FBS was added to each well, and antibodies produced at twice (2Х) the final concentration (100 nM) were serially diluted 1 / 10 and added to the wells in 100 μl at 6 concentrations for each antibody (i.e., 200 nM, 20 nM, 2 nM, 200 pM, 20 pM, and 2 pM). Then, the plate was cultured for 5 days at 37℃, 95% relative humidity, and 5% (v / v) CO2, and on the last day, the cells were fixed with 10% trichloroacetic acid (TCA; Sigma, #T0699) solution. The fixed cells were stained for 25 minutes by adding 80 μl of 0.4% sulforhodamine B (SRB) solution to each well, followed by five washes with 1% acetic acid solution. Then, 150 μl of 10 mM Tris solution was added to each well of the dried plate to dissolve the SRB dye, and the absorbance was measured at a wavelength of 540 nm using a microplate reader.

[0179] In addition, the MKN45 (#JCRB0254) cell line was diluted in RPMI-1640 medium (Gibco, #A10491) containing 10% (v / v) FBS, seeded at 2.5 Х 103into each well of a 96-well plate, and incubated overnight under 37℃ and 5% CO2conditions. The medium in each well of the plate was then replaced with 100 ㎕ of RPMI-1640 medium containing 1% (v / v) FBS, and the test antibody was sequentially diluted 1 / 10 from a final concentration of 100 nM to 1 pM (i.e., 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM) and added in 100 μl to each well. Then, the plate was cultured at 37℃ and 5% CO2for 5 days, the medium was removed, and 200 μl of TCA solution was added to each well for cell fixation. Similar to the test for U87 MG cells, the cells in the plate were stained according to a typical SRB colorimetric assay method, and the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader. The results for the U87 MG and MKN45 cell lines are shown in Table 2.

[0180]

[0181] U-87 MG(GBM, HGF autocrine)IC50(nM)MKN45(Gastric cancer, c-MET amplified)IC50(nM)LY2875358(Eli Lilly)> 1000.34OA-5D5(Genentech)> 100> 100mCM0517.59.78cCM05-IgG132.4> 100cCM05-IgG2> 10012.92

[0182]

[0183] As shown in Table 2, the anti-c-MET antibodies of the present disclosure all have comparable or better tumor cell proliferation inhibitory activity compared to the known c-MET antibodies LY2875358 and OA-5D5 (control groups).

[0184] The specific consensus sequences for the light chain and heavy chain variable regions of the antibodies of the present disclosure are shown in Table 3 below.

[0185]

[0186] Amino acid consensus sequenceNucleotide consensus sequenceLight chainHeavy chainLight chainHeavy chainDILMTQSPASLSASVGETVTITCGASENIYGALNWYQRKQGKSPQLLIYGATNLADGMSSRFSGSGSGRQFSLKITSLHPDDVATYYCQNVLSSPYTFGGGTKLEIK (SEQ ID NO: 7)EVQLQQSGAELARPGASVKLSCKASGYTFSDYYINWVKQGTGQGLEWIGEIFPGSGNTHFSARFKGKATLTADKSSSTAYMQLSSLTSTDSAVYFCAGGDYGFLYWGRGTLVTVSA (SEQ ID NO: 8)gatattctgatgacccagtctccagcttcactgtctgcatctgtgggagaaactgtcaccatcacatgtggagcaagtgagaatatttacggtgctttaaattggtatcagcgaaaacagggaaaatctcctcagctcctgatctatggtgcaaccaacttggcagatggcatgtcatcgaggttcagtggcagtgggtctggtagacagttttctctcaagatcactagcctgcatcctgacgatgttgcaacgtattactgtcaaaatgtgctaagtagtccgtacacgttcggaggggggaccaagctggaaatcaaa (SEQ ID NO: 9)gaggttcagctgcagcagtctggagctgagctggcgaggcccggggcttcagtgaagctgtcctgcaaggcttctggctacaccttcagtgactactatataaactgggtgaagcaggggactggacagggccttgagtggattggagagatttttcctggaagtggaaatactcacttcagtgcgaggttcaagggcaaggccacactgactgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctacggactctgcagtctatttctgtgccgggggtgactacgggtttctttactggggccgagggactctggtcactgtctctgca (SEQ ID NO: 10)

[0187]

[0188] Example 2. Preparation of humanized antibody from mCM05 antibody and confirmation of in vitro tumor cell proliferation inhibitory activity thereof

[0189] To further confirm the effect of the antibodies produced in the present disclosure, as an example, the mouse antibody mCM05 was humanized, and in vitro tumor cell proliferation inhibitory activity thereof was confirmed.

[0190] For the humanized design of the mCM05 antibody heavy chain, first, human germline gene which has high homology to the heavy chain variable region gene of the mCM05 antibody was analyzed by Ig Blast (http: / www.ncbi.nlm.nih.gov / igblast / ). As a result, it was confirmed that IGHV3-23 has 48% homology with the mCM05 antibody at the amino acid level, and IGHV3-11 has 46% homology with the mCM05 antibody at the amino acid level.

[0191] CDR-H1, CDR-H2, and CDR-H3 of the mCM05 antibody were defined by Kabat numbering, and the CDR portion of the mCM05 antibody was designed to be introduced into the framework of IGHV3-23 to produce huCM05-1. Here, amino acid Nos. 48 (V→I), 49 (S→G), 71 (R→A), 73 (N→K), 78 (L→A), and 94 (K→G) were subjected to back-mutation to the original amino acid sequence of the mCM05 antibody to finally construct heavy chain of huCM05-1. For huCM05-2, the CDR portion of the mCM05 antibody was designed to be introduced into the framework of IGHV3-11, and the amino acid Nos. 48 (V→I), 49 (S→G), 71 (R→A), 73 (N→K), 78 (L→A), and 94 (R→G) were subjected to back-mutation to the original amino acid sequence of the mCM05 antibody to finally construct heavy chain of huCM05-2.

[0192] For the mCM05 antibody light chain, human germline gene which has high homology to the gene of the light chain variable region of the mCM05 antibody was analyzed by Ig Blast for humanization design. As a result, it was confirmed that IGKV1-27 has 65.3 % homology with the mCM05 antibody at the amino acid level, and IGKV1-33 has 64.2 % homology with the mCM05 antibody at the amino acid level.

[0193] CDR-L1, CDR-L2, and CDR-L3 of the mCM05 antibody were defined by Kabat numbering, and the CDR portion of the mCM05 antibody was designed to be introduced into the framework of IGKV1-33 to produce huCM05-1, and was designed to be introduced into the framework of IGKV1-27 to produce huCM05-2. Here, amino acid No. 69 (T→R) of both huCM05-1 and huCM05-2 was back-mutated to the original amino acid sequence of the mCM05 antibody.

[0194] The CM05 humanized antibody was expressed in 293T cells using the pCLS05 vector (Korean Patent No. 10-1420274). The humanized antibodies thus obtained were tested for tumor cell proliferation inhibitory activity in U87 MG, a human glioblastoma cell line, in the same manner as in Example 1 above.

[0195] As a result, it was confirmed that the IC50values of huCM05-1 and huCM05-2 were 30 nM and 24.6 nM, respectively, indicating similar levels of anticancer activity to the chimeric cCM05-IgG1 antibody (IC50= 32.4 nM).

[0196] The specific consensus sequences of the light chain and heavy chain variable regions of the huCM05-1 and huCM05-2 humanized antibodies are shown in Table 4.

[0197]

[0198] Amino acid consensus sequenceNucleotide consensus sequenceLight chainHeavy chainLight chainHeavy chainhuCM05-1DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLSSPYTFGQGTKVEIK (SEQ ID NO: 11)EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYYINWVRQAPGKGLEWIGEIFPGSGNTHFSARFKGRATLSADKSKNTAYLQMNSLRAEDTAVYYCAGGDYGFLYWGQGTLVTVSS (SEQ ID NO: 12)gatatccagatgacccagtctcccagcagtctttccgcttctgtgggtgatagggtgacgataacttgcggagcaagtgagaatatttacggtgctttaaattggtaccagcagaagcctgggaaagctccaaagctgctgatctatggtgcaaccaacttggcagatggcgtccctagcaggttcagcggcagtggaagcggcagagacttcactttcacaatctcctccctgcaacccgaggacattgcaacctactattgtcaaaatgtgctaagtagtccgtacacgtttggccagggaaccaaggttgaaattaaa (SEQ ID NO: 13)gaggttcagttagtggaatccggaggaggactggtgcagcctggtggaagtttgaggctgtcatgcgcagccagtggctacaccttcagtgactactatataaactgggtaagacaggctcccggaaaagggctggagtggattggagagatttttcctggaagtggaaatactcacttcagtgcgaggttcaagggccgagccaccctctcagcagacaaaagcaagaataccgcctatctgcagatgaatagccttcgcgcagaagatactgccgtgtattactgtgccgggggtgactacgggtttctttactggggacagggcaccttggtgacagtctcttct (SEQ ID NO: 14)huCM05-2DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKVPKLLIYGATNLADGVPSRFSGSGSGRDFTLTISSLQPEDVATYYCQNVLSSPYTFGQGTKVEIK (SEQ ID NO: 15)QVQLVESGGGLVKPGGSLRLSCAASGYTFSDYYINWIRQAPGKGLEWIGEIFPGSGNTHFSARFKGRATISADKAKNSAYLQMNSLRAEDTAVYYCAGGDYGFLYWGQGTLVTVSS (SEQ ID NO: 16)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcggagcaagtgagaatatttacggtgctttaaattggtatcagcagaaaccagggaaagttcctaagctcctgatctatggtgcaaccaacttggcagatggggtcccatctcggttcagtggcagtggatctgggcgagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaatgtgctaagtagtccgtacacgtttggccagggaaccaaggttgaaattaaa (SEQ ID NO: 17)caggttcagttagtggaatccggaggaggactggtgaagcctggtggaagtttgaggctgtcatgcgcagccagtggctacaccttcagtgactactatataaactggatcagacaggctcccggaaaagggctggagtggattggagagatttttcctggaagtggaaatactcacttcagtgcgaggttcaagggccgagccaccatctcagcagacaaagcgaagaatagcgcctatctgcagatgaatagccttcgcgcagaagatactgccgtgtattactgtgccgggggtgactacgggtttctttactggggacagggcaccttggtgacagtctcttct (SEQ ID NO: 18)

[0199]

[0200] Example 3. Preparation of hinge mutants and test of tumor cell proliferation inhibitory activity thereof

[0201] Next, tumor cell proliferation inhibitory activity test was performed according to the hinge sequence of the human IgG1 heavy chain constant region.

[0202] First, the hinge of the human IgG1 heavy chain constant region, which has the amino acid sequence 'EPKSCDKTHTCPPCP (SEQ ID NO: 19)', was substituted to obtain hinge region mutants having the amino acid sequence of SEQ ID NO: 20 to SEQ ID NO: 26. The hinge region mutant was cloned into vectors containing the heavy chain variable regions of the huCM05-1 and huCM05-2 humanized antibodies produced in Example 2 above, respectively. In vitro tumor cell proliferation inhibitory activity according to the hinge sequence was confirmed in U-87 MG in the same manner as in Example 1 above.

[0203] In addition, the effect of huCM05-1 and huCM05-2 humanized antibodies on non-small cell lung cancer cell line NCI-H1993 (ATCC, #CRL-5909) was analyzed as follows. The NCI-H1993 cell line was diluted in RPMI-1640 medium (Gibco, #A10491) containing 10% (v / v) FBS, seeded at 3.0 Х 103into each well of a 96-well plate, and incubated overnight under 37℃ and 5% CO2conditions. The medium in each well of the plate was then replaced with 100 μl of RPMI-1640 medium containing 2 % (v / v) FBS, and the test antibody was sequentially diluted 1 / 10 from a final concentration of 100 nM to 0.001 nM (i.e., 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM) and added in 100 μl to each well. Then, the plate was cultured at 37℃ and 5% CO2for 5 days, the medium was removed, and 200 μl of TCA solution (Sigma, #T0699) was added to each well for cell fixation. Cells on the plate were stained according to a typical SRB colorimetric assay method, and the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader.

[0204] The results of huCM05-1 in U-87 MG and NCI-H1993 (ATCC, #CRL-5909) are shown in Table 5.

[0205]

[0206] Hinge region amino acid sequenceHinge region nucleotide sequenceU-87 MG(GBM, HFG autocrine)IC50nMNCI-H1993(NSCLC,c-MET amplified)IC50nMEPKSCDKTHTCPPCP (SEQ ID NO: 19)gagcccaaatcttgtgacaaaactcacacatgcccaccgtgccca (SEQ ID NO: 27)12.6> 100ERKCCVECPPCP (SEQ ID NO: 20)gagcgaaaatgttgtgtcgagtgcccaccgtgccca (SEQ ID NO: 28)31.00.30ECCVECPPCP(SEQ ID NO: 21)gagtgttgtgtcgagtgcccaccgtgccca (SEQ ID NO: 29)57.3> 100ERKCCCPPCP(SEQ ID NO: 22)gagcgaaaatgttgttgcccaccgtgccca (SEQ ID NO: 30)37.60.23ECCCPPCP(SEQ ID NO: 23)gagtgttgttgcccaccgtgccca (SEQ ID NO: 31)25.3> 100EKCCVECPPCP(SEQ ID NO: 24)gagaaatgttgtgtcgagtgcccaccgtgccca (SEQ ID NO: 32)31.40.48ERKCCVCPPCP(SEQ ID NO: 25)gagcgaaaatgttgtgtctgcccaccgtgccca (SEQ ID NO: 33)30.80.47EKCCVCPPCP(SEQ ID NO: 26)gagaaatgttgtgtctgcccaccgtgccca (SEQ ID NO: 34)75.90.38

[0207]

[0208] As shown in Table 5, the anti-tumor cell proliferation activity of huCM05-1 antibodies varied somewhat depending on the difference in hinge sequence, but it was confirmed that most of them inhibited the proliferation of tumor cells effectively. Accordingly, hereinafter, an IgG1 humanized antibody having a hinge region of SEQ ID NO: 20 in huCM05-1 is representatively named huCM05, and an affinity-optimized antibody for huCM05 was prepared to confirm its effect.

[0209]

[0210] Example 4. Preparation of affinity-optimized antibody for huCM05 and confirmation of in vitro tumor cell proliferation inhibitory activity thereof

[0211] To prepare an affinity-optimized antibody for huCM05, a phage-displayed scFv library was first prepared using a phagemid vector in which scFv and pIII were displayed in a combined form, and the schematic structure of the vector is shown in FIG. 1. The phagemid vector contained the scFv fragment of the antibody under the control of the IPTG-inducible lac promoter, and the linker sequence used was GGGGS GGGGS GGGGS (SEQ ID NO: 35).

[0212] Next, mutagenic oligonucleotides with NNK codons were used to introduce diversity into the heavy and light chain CDR regions of huCM05. Accordingly, a huCM05 scFv library fused with His, HA, and pIII was prepared, and antibodies specific to human c-MET were selected from the prepared antibody library.

[0213] Specifically, a competitive selection method was used to select antibodies with improved affinity. Human c-MET antigen was bound to Dynabeads® M-280 (Thermo Fisher Scientific, 11205D) according to the manufacturer's instructions. Antigen-bound beads were blocked with Superblock Tris buffered saline (Superblock TBS, Pierce) for 2 hours. In addition, the recombinant phage was grown overnight at 37℃, centrifuged, and the phage in the supernatant was blocked with Superblock TBS and 0.05% Tween 20 for 2 hours. The beads were then washed with PBS containing 0.05% Tween 20. The blocked phage solution was added to the washed beads, incubated for 2 hours in a rotator for phage binding, and the beads were washed with PBS containing 0.05% Tween 20. Next, human c-MET antigen was added to 1 ml of PBS containing 0.05% Tween 20 and incubated in a rotator for 24 hours (Rouet R et al. (2012) Nat Protoc. 7:364-373). Next, phage bound to the beads was eluted with 100 mM triethanolamine for 5 minutes, and the eluate was neutralized with 0.5 M Tris / Cl (pH 7.2). The eluted phage solution which was neutralized was transfected with E. coli TG1.

[0214] Individual clones selected from the above experiments were grown in a 96-well format, using 200 μl of 2xYT broth supplemented with carbenicillin and ampicillin, and culture supernatants were directly employed in ELISA to screen for phage-displayed scFv that bind to a plate coated with target proteins. The amino acid sequences of the light and heavy chain CDR regions of the detected antibodies are shown in Tables 6 and 7, and the representative amino acid sequences of the variable regions of light chain and heavy chain affinity-optimized antibodies are shown in Table 8.

[0215]

[0216] Heavy chain CDR sequence listingCDR1CDR2CDR3AH01DYYIN (SEQ ID NO: 4)EIDPGSGNTHFSARFKG (SEQ ID NO: 41)GDYGFLY (SEQ ID NO: 6)AH02DYYIN (SEQ ID NO: 4)EIEPGSGNTHFSARFKG (SEQ ID NO: 42)GDYGFLY (SEQ ID NO: 6)AH03DYYIN (SEQ ID NO: 4)EIWPGSGNTHFSARFKG (SEQ ID NO: 43)GDYGFLY (SEQ ID NO: 6)AH04DYYIN (SEQ ID NO: 4)EIYPGSGNTHFSARFKG (SEQ ID NO: 44)GDYGFLY (SEQ ID NO: 6)AH05DYYIN (SEQ ID NO: 4)EIFPGWGNTHFSARFKG (SEQ ID NO: 45)GDYGFLY (SEQ ID NO: 6)AH06DYYIN (SEQ ID NO: 4)EIFPGYGNTHFSARFKG (SEQ ID NO: 46)GDYGFLY (SEQ ID NO: 6)AH07DYYIN (SEQ ID NO: 4)EIFPGSGYTHFSARFKG (SEQ ID NO: 47)GDYGFLY (SEQ ID NO: 6)AH08DYYIN (SEQ ID NO: 4)EIFPGSGNTWFSARFKG (SEQ ID NO: 48)GDYGFLY (SEQ ID NO: 6)AH09DYYIN (SEQ ID NO: 4)EIFPGSGNTYFSARFKG (SEQ ID NO: 49)GDYGFLY (SEQ ID NO: 6)AH12DYYIN (SEQ ID NO: 4)EIFPGWGNTYFSARFKG (SEQ ID NO: 50)GDYGFLY (SEQ ID NO: 6)AH13DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLY (SEQ ID NO: 111)AH14DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)EDYGFLY (SEQ ID NO: 112)AH15DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)HDYGFLY (SEQ ID NO: 113)AH16DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)NDYGFLY (SEQ ID NO: 114)AH17DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)VELGFLY (SEQ ID NO: 115)AH18DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)FETGYYL (SEQ ID NO: 116)AH19DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GEYGYQN (SEQ ID NO: 117)AH20DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)WEYGLSM (SEQ ID NO: 118)AH21DYYIN (SEQ ID NO: 4)EIFPHFTSDHFSARFKG (SEQ ID NO: 51)GDYGFLY (SEQ ID NO: 6)AH22DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSAWMGT (SEQ ID NO: 52)GDYGFLY (SEQ ID NO: 6)AH23DYYIN (SEQ ID NO: 4)EIFPGSGNESVSFRFKG (SEQ ID NO: 53)GDYGFLY (SEQ ID NO: 6)AH24DYYIN (SEQ ID NO: 4)EIFPGSGNSAVISRFKG (SEQ ID NO: 54)GDYGFLY (SEQ ID NO: 6)AH25DYYIN (SEQ ID NO: 4)EIFPGSGNHTVVRRFKG (SEQ ID NO: 55)GDYGFLY (SEQ ID NO: 6)AH26DYYIN (SEQ ID NO: 4)EIFPGSGNLSMHGRFKG (SEQ ID NO: 56)GDYGFLY (SEQ ID NO: 6)AH27DYYIN (SEQ ID NO: 4)EIFPGSGNHTPVFRFKG (SEQ ID NO: 57)GDYGFLY (SEQ ID NO: 6)AH28DYYIN (SEQ ID NO: 4)EIFPGSGNPFLTIRFKG (SEQ ID NO: 58)GDYGFLY (SEQ ID NO: 6)AH29DYYIN (SEQ ID NO: 4)EIFPGSGNSHVVSRFKG (SEQ ID NO: 59)GDYGFLY (SEQ ID NO: 6)AH30DYYIN (SEQ ID NO: 4)EIFPGSGNLSGIRSFKG (SEQ ID NO: 60)GDYGFLY (SEQ ID NO: 6)AH31DYYIN (SEQ ID NO: 4)EIFPGSGNFFHGKRFKG (SEQ ID NO: 61)GDYGFLY (SEQ ID NO: 6)AH32DYYIN (SEQ ID NO: 4)EIFPGSGNPRLGARFKG (SEQ ID NO: 62)GDYGFLY (SEQ ID NO: 6)AH33DYYIN (SEQ ID NO: 4)EIFPGSGNVSQVERFKG (SEQ ID NO: 63)GDYGFLY (SEQ ID NO: 6)AH34DYYIN (SEQ ID NO: 4)EIFPGSGNFHGASRFKG (SEQ ID NO: 64)GDYGFLY (SEQ ID NO: 6)AH35DYYIN (SEQ ID NO: 4)EIFPGSGNVVGGYRFKG (SEQ ID NO: 65)GDYGFLY (SEQ ID NO: 6)AH36DYYIN (SEQ ID NO: 4)EIFPGSGNPMYDERFKG (SEQ ID NO: 66)GDYGFLY (SEQ ID NO: 6)AH37DYYIN (SEQ ID NO: 4)EIFPGSGNADLTIRFKG (SEQ ID NO: 67)GDYGFLY (SEQ ID NO: 6)AH38DYYIN (SEQ ID NO: 4)EIFPGSGNSTNLYRFKG (SEQ ID NO: 68)GDYGFLY (SEQ ID NO: 6)AH39DYYIN (SEQ ID NO: 4)EIFPGSGNLDIPPRFKG (SEQ ID NO: 69)GDYGFLY (SEQ ID NO: 6)AH40DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSSAPLP (SEQ ID NO: 70)GDYGFLY (SEQ ID NO: 6)AH41DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSSEFVS (SEQ ID NO: 71)GDYGFLY (SEQ ID NO: 6)AH42DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSMSESF (SEQ ID NO: 72)GDYGFLY (SEQ ID NO: 6)AH43DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSDGSRN (SEQ ID NO: 73)GDYGFLY (SEQ ID NO: 6)AH44DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSSSVSR (SEQ ID NO: 74)GDYGFLY (SEQ ID NO: 6)AH45DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSRSVSG (SEQ ID NO: 75)GDYGFLY (SEQ ID NO: 6)AH46DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSGLSEV (SEQ ID NO: 76)GDYGFLY (SEQ ID NO: 6)AH47DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSHYWAS (SEQ ID NO: 77)GDYGFLY (SEQ ID NO: 6)AH48DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSTGLTQ (SEQ ID NO: 78)GDYGFLY (SEQ ID NO: 6)AH49DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSRHRLH (SEQ ID NO: 79)GDYGFLY (SEQ ID NO: 6)AH50DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSVPRSM (SEQ ID NO: 80)GDYGFLY (SEQ ID NO: 6)AH51DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSLQDYL (SEQ ID NO: 81)GDYGFLY (SEQ ID NO: 6)AH52DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSDGVSS (SEQ ID NO: 82)GDYGFLY (SEQ ID NO: 6)AH53DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSMQGSE (SEQ ID NO: 83)GDYGFLY (SEQ ID NO: 6)AH54DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSGNVHW (SEQ ID NO: 84)GDYGFLY (SEQ ID NO: 6)AH55DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSRSPTP (SEQ ID NO: 85)GDYGFLY (SEQ ID NO: 6)AH56DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSLRMFP (SEQ ID NO: 86)GDYGFLY (SEQ ID NO: 6)AH57DYYAN (SEQ ID NO: 36)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLY (SEQ ID NO: 6)AH58GYYIN (SEQ ID NO: 37)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLY (SEQ ID NO: 6)AH59QYYIN (SEQ ID NO: 38)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLY (SEQ ID NO: 6)AH60DQYIN (SEQ ID NO: 39)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLY (SEQ ID NO: 6)AH61DYYQN (SEQ ID NO: 40)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLY (SEQ ID NO: 6)AH62DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDVGFLY (SEQ ID NO: 119)AH63DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFQY (SEQ ID NO: 120)AH64DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDYGFLQ (SEQ ID NO: 121)AH65DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)GDQWLLC (SEQ ID NO: 122)AH66DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSARFKG (SEQ ID NO: 5)WDYGFLY (SEQ ID NO: 123)AH67DYYIN (SEQ ID NO: 4)EIFPDSAPSHFSARFKG (SEQ ID NO: 87)GDYGFLY (SEQ ID NO: 6)AH68DYYIN (SEQ ID NO: 4)EIFPYFLPPHFSARFKG (SEQ ID NO: 88)GDYGFLY (SEQ ID NO: 6)AH69DYYIN (SEQ ID NO: 4)EIFPGPFTPHFSARFKG (SEQ ID NO: 89)GDYGFLY (SEQ ID NO: 6)AH70DYYIN (SEQ ID NO: 4)EIFPGSNFGHFSARFKG (SEQ ID NO: 90)GDYGFLY (SEQ ID NO: 6)AH71DYYIN (SEQ ID NO: 4)EIFPGWGNTHFSARFKG (SEQ ID NO: 45)GDYGFLY (SEQ ID NO: 111)AH72DYYIN (SEQ ID NO: 4)EIFPGWGNTHFSRSPTP (SEQ ID NO: 91)GDYGFLY (SEQ ID NO: 6)AH73DYYIN (SEQ ID NO: 4)EIFPGWGNSHVVSRFKG (SEQ ID NO: 92)GDYGFLY (SEQ ID NO: 6)AH74DYYIN (SEQ ID NO: 4)EIFPGYGNTHFSARFKG (SEQ ID NO: 46)GDYGFLY (SEQ ID NO: 111)AH75DYYIN (SEQ ID NO: 4)EIFPGYGNTYFSARFKG (SEQ ID NO: 93)GDYGFLY (SEQ ID NO: 6)AH76DYYIN (SEQ ID NO: 4)EIFPGYGNTHFSRSPTP (SEQ ID NO: 94)GDYGFLY (SEQ ID NO: 6)AH77DYYIN (SEQ ID NO: 4)EIFPGYGNSHVVSRFKG (SEQ ID NO: 95)GDYGFLY (SEQ ID NO: 6)AH78DYYIN (SEQ ID NO: 4)EIFPGSGNTYFSARFKG (SEQ ID NO: 49)GDYGFLY (SEQ ID NO: 111)AH79DYYIN (SEQ ID NO: 4)EIFPGSGNTYFSRSPTP (SEQ ID NO: 96)GDYGFLY (SEQ ID NO: 6)AH80DYYIN (SEQ ID NO: 4)EIFPGSGNSHVVSRFKG (SEQ ID NO: 59)GDYGFLY (SEQ ID NO: 111)AH81DYYIN (SEQ ID NO: 4)EIFPGSGNSHVVRSPTP (SEQ ID NO: 97)GDYGFLY (SEQ ID NO: 6)AH82DYYIN (SEQ ID NO: 4)EIFPGSGNSHVVRSPTP (SEQ ID NO: 97)GDYGFLY (SEQ ID NO: 6)AH83DYYIN (SEQ ID NO: 4)EIFPGWGNTYFSARFKG (SEQ ID NO: 50)GDYGFLY (SEQ ID NO: 111)AH84DYYIN (SEQ ID NO: 4)EIFPGWGNTHFSRSPTP (SEQ ID NO: 91)GDYGFLY (SEQ ID NO: 111)AH85DYYIN (SEQ ID NO: 4)EIFPGWGNSHVVSRFKG (SEQ ID NO: 92)GDYGFLY (SEQ ID NO: 111)AH86DYYIN (SEQ ID NO: 4)EIFPGYGNTYFSARFKG (SEQ ID NO: 93)GDYGFLY (SEQ ID NO: 111)AH87DYYIN (SEQ ID NO: 4)EIFPGYGNSHVVSRFKG (SEQ ID NO: 95)GDYGFLY (SEQ ID NO: 111)AH88DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSRSPTP (SEQ ID NO: 85)GDYGFLY (SEQ ID NO: 111)AH89DYYIN (SEQ ID NO: 4)EIFPGYGNTHFSRSPTP (SEQ ID NO: 94)GDYGFLY (SEQ ID NO: 111)AH90DYYIN (SEQ ID NO: 4)EIFPGSGNTYFSRSPTP (SEQ ID NO: 96)GDYGFLY (SEQ ID NO: 111)AH91DYYIN (SEQ ID NO: 4)EIFPGSGNSHVVRSPTP (SEQ ID NO: 97)GDYGFLY (SEQ ID NO: 111)AH92DYYIN (SEQ ID NO: 4)EIFPGSGNSHVVSSPTP (SEQ ID NO: 98)GDYGFLY (SEQ ID NO: 111)AH93DYYIN (SEQ ID NO: 4)EIFPDSAPSYFSARFKG (SEQ ID NO: 99)GDYGFLY (SEQ ID NO: 6)AH94DYYIN (SEQ ID NO: 4)EIFPGPFTPYFSARFKG (SEQ ID NO: 100)GDYGFLY (SEQ ID NO: 6)AH95DYYIN (SEQ ID NO: 4)EIFPGSNFGYFSRSPTP (SEQ ID NO: 101)GDYGFLY (SEQ ID NO: 6)AH96DYYIN (SEQ ID NO: 4)EIFPDSAPSHVVSRFKG (SEQ ID NO: 102)GDYGFLY (SEQ ID NO: 6)AH97DYYIN (SEQ ID NO: 4)EIFPGPFTSHVVSRFKG (SEQ ID NO: 103)GDYGFLY (SEQ ID NO: 6)AH98DYYIN (SEQ ID NO: 4)EIFPGSNFSHVVSRFKG (SEQ ID NO: 104)GDYGFLY (SEQ ID NO: 6)AH99DYYIN (SEQ ID NO: 4)EIFPDSAPSHFSRSPTP (SEQ ID NO: 105)GDYGFLY (SEQ ID NO: 6)AH100DYYIN (SEQ ID NO: 4)EIFPGPFTPHFSRSPTP (SEQ ID NO: 106)GDYGFLY (SEQ ID NO: 6)AH101DYYIN (SEQ ID NO: 4)EIFPGSNFGHFSRSPTP (SEQ ID NO: 107)GDYGFLY (SEQ ID NO: 6)AH102DYYIN (SEQ ID NO: 4)EIFPDSAPSHVVSSPTP (SEQ ID NO: 108)GDYGFLY (SEQ ID NO: 6)AH103DYYIN (SEQ ID NO: 4)EIFPGPFTSHVVSSPTP (SEQ ID NO: 109)GDYGFLY (SEQ ID NO: 6)AH104DYYIN (SEQ ID NO: 4)EIFPGSNFSHVVSSPTP (SEQ ID NO: 110)GDYGFLY (SEQ ID NO: 6)AH105QYYIN (SEQ ID NO: 38)EIFPDSAPSHFSARFKG (SEQ ID NO: 87)GDYGFLY (SEQ ID NO: 6)AH106QYYIN (SEQ ID NO: 38)EIFPGPFTPHFSARFKG (SEQ ID NO: 89)GDYGFLY (SEQ ID NO: 6)AH107QYYIN (SEQ ID NO: 38)EIFPGSNFGHFSARFKG (SEQ ID NO: 90)GDYGFLY (SEQ ID NO: 6)AH108DYYIN (SEQ ID NO: 4)EIFPDSAPSHFSARFKG (SEQ ID NO: 87)GDYGFLY (SEQ ID NO: 111)AH109DYYIN (SEQ ID NO: 4)EIFPGPFTPHFSARFKG (SEQ ID NO: 89)GDYGFLY (SEQ ID NO: 111)AH110DYYIN (SEQ ID NO: 4)EIFPGSNFGHFSARFKG (SEQ ID NO: 90)GDYGFLY (SEQ ID NO: 111)AH111DYYIN (SEQ ID NO: 4)EIFPDSAPSHFSARFKG (SEQ ID NO: 87)GDYGFQY (SEQ ID NO: 120)AH112DYYIN (SEQ ID NO: 4)EIFPGPFTPHFSARFKG (SEQ ID NO: 89)GDYGFQY (SEQ ID NO: 120)AH113DYYIN (SEQ ID NO: 4)EIFPGSNFGHFSARFKG (SEQ ID NO: 90)GDYGFQY (SEQ ID NO: 120)AH114DYYIN (SEQ ID NO: 4)EIFPDSAPSHFSARFKG (SEQ ID NO: 87)GDYGFLQ (SEQ ID NO: 121)AH115DYYIN (SEQ ID NO: 4)EIFPGPFTPHFSARFKG (SEQ ID NO: 89)GDYGFLQ (SEQ ID NO: 121)AH116DYYIN (SEQ ID NO: 4)EIFPGSNFGHFSARFKG (SEQ ID NO: 90)GDYGFLQ (SEQ ID NO: 121)AH117DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSMSESF (SEQ ID NO: 72)HDYGFLY (SEQ ID NO: 113)AH118DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSLQDYL (SEQ ID NO: 81)HDYGFLY (SEQ ID NO: 113)AH119DYYIN (SEQ ID NO: 4)EIFPGSGNTHFSMQGSE (SEQ ID NO: 83)HDYGFLY (SEQ ID NO: 113)

[0217]

[0218] Light chain CDR sequence listingCDR1CDR2CDR3AL01GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVWSSPYT (SEQ ID NO: 175)AL02GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLNSPYT (SEQ ID NO: 176)AL03GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLESPYT (SEQ ID NO: 177)AL04GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLKSPYT (SEQ ID NO: 178)AL05GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLYSPYT (SEQ ID NO: 179)AL06GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSRPYT (SEQ ID NO: 180)AL07GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSSPET (SEQ ID NO: 181)AL08GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSEPYT (SEQ ID NO: 182)AL11GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLESPET (SEQ ID NO: 183)AL12GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSVPET (SEQ ID NO: 184)AL13GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSLPET (SEQ ID NO: 185)AL14GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSIPET (SEQ ID NO: 186)AL15GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSMPET (SEQ ID NO: 187)AL16GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILSSPET (SEQ ID NO: 188)AL17GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLISSPET (SEQ ID NO: 189)AL18GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNMISSPET (SEQ ID NO: 190)AL19GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNIISLPET (SEQ ID NO: 191)AL20GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNIISIPET (SEQ ID NO: 192)AL21GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNSLSSPET (SEQ ID NO: 193)AL22GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNTLSSPET (SEQ ID NO: 194)AL23GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVSSSPET (SEQ ID NO: 195)AL24GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVISSPET (SEQ ID NO: 196)AL25GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVFSSPET (SEQ ID NO: 197)AL26GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVYSSPET (SEQ ID NO: 198)AL27GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVRSSPET (SEQ ID NO: 199)AL28GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLVSSPET (SEQ ID NO: 200)AL29GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLISSPET (SEQ ID NO: 201)AL30GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLMSSPET (SEQ ID NO: 202)AL31GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNIMSSPET (SEQ ID NO: 203)AL32GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVHSSPET (SEQ ID NO: 204)AL33GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVMSSPET (SEQ ID NO: 205)AL34GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLSSPET (SEQ ID NO: 206)AL35GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QSVLFSPFS (SEQ ID NO: 207)AL36GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QQVLFFPET (SEQ ID NO: 208)AL37GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLSPSFY (SEQ ID NO: 209)AL38GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QSVLFSPFT (SEQ ID NO: 210)AL39GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILSSPLF (SEQ ID NO: 211)AL40GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNTLHYSLV (SEQ ID NO: 212)AL41GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QQVLFFPLL (SEQ ID NO: 213)AL42GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QQVLDFVFY (SEQ ID NO: 214)AL43GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVVSSPET (SEQ ID NO: 215)AL44GASENIYGALN (SEQ ID NO: 1)DATNLAD (SEQ ID NO: 164)QNVLSSPYT (SEQ ID NO: 3)AL45GASENIYGALN (SEQ ID NO: 1)FATNLAD (SEQ ID NO: 165)QNVLSSPYT (SEQ ID NO: 3)AL46GASENIYGALN (SEQ ID NO: 1)HATNLAD (SEQ ID NO: 166)QNVLSSPYT (SEQ ID NO: 3)AL47GASENIYGALN (SEQ ID NO: 1)KATNLAD (SEQ ID NO: 167)QNVLSSPYT (SEQ ID NO: 3)AL48GASENIYGALN (SEQ ID NO: 1)PATNLAD (SEQ ID NO: 168)QNVLSSPYT (SEQ ID NO: 3)AL49GASENIYGALN (SEQ ID NO: 1)QATNLAD (SEQ ID NO: 169)QNVLSSPYT (SEQ ID NO: 3)AL50GASENIYGALN (SEQ ID NO: 1)SATNLAD (SEQ ID NO: 170)QNVLSSPYT (SEQ ID NO: 3)AL51GASENIYGALN (SEQ ID NO: 1)VATNLAD (SEQ ID NO: 171)QNVLSSPYT (SEQ ID NO: 3)AL52GASENIYGALN (SEQ ID NO: 1)YATNLAD (SEQ ID NO: 172)QNVLSSPYT (SEQ ID NO: 3)AL53GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)ITVLSPPYT (SEQ ID NO: 216)AL54GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNNLVPPFN (SEQ ID NO: 217)AL55GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QHVLFLPYV (SEQ ID NO: 218)AL56GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QAVLTNAYT (SEQ ID NO: 219)AL57GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLRVGYL (SEQ ID NO: 220)AL58GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QSVLRVGYL (SEQ ID NO: 221)AL59GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNIISSPYT (SEQ ID NO: 222)AL60GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QQVLCESFL (SEQ ID NO: 223)AL61GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSQSLL (SEQ ID NO: 224)AL62GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLQPSYL (SEQ ID NO: 225)AL63GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLFQPLS (SEQ ID NO: 226)AL64GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLFQPLV (SEQ ID NO: 227)AL65GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNQLDPSLF (SEQ ID NO: 228)AL66GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)MDVLESPYT (SEQ ID NO: 229)AL67GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QALLLSPYT (SEQ ID NO: 230)AL68GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QQLLESPYT (SEQ ID NO: 231)AL69GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)NLTLVSPYT (SEQ ID NO: 232)AL70GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)GNILDSPYT (SEQ ID NO: 233)AL71GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)EQVLLSPYT (SEQ ID NO: 234)AL72GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)NNLLDSPYT (SEQ ID NO: 235)AL73GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)EEVLSSPYT (SEQ ID NO: 236)AL74GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILFVDYT (SEQ ID NO: 237)AL75GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLHLNYT (SEQ ID NO: 238)AL76GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLQTPYT (SEQ ID NO: 239)AL77GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILHPGYT (SEQ ID NO: 240)AL78GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLTRGYT (SEQ ID NO: 241)AL79GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)ENILYSPYT (SEQ ID NO: 242)AL80GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLGGGQG (SEQ ID NO: 243)AL81GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLEHPLI (SEQ ID NO: 244)AL82GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLDDPFD (SEQ ID NO: 245)AL83GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLDFPLL (SEQ ID NO: 246)AL84GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLYPSLV (SEQ ID NO: 247)AL85GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLFDQQS (SEQ ID NO: 248)AL86GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSNEET (SEQ ID NO: 249)AL87GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLKHPYT (SEQ ID NO: 250)AL88GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSPGMW (SEQ ID NO: 251)AL89GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLSSPYT (SEQ ID NO: 3)AL90GASENIYGALN (SEQ ID NO: 1)GAQNLAD (SEQ ID NO: 174)QNVLSSPYT (SEQ ID NO: 3)AL91GSSRSIYGALN (SEQ ID NO: 124)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL92RAGRSIYGALN (SEQ ID NO: 125)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL93LGRRGIYGALN (SEQ ID NO: 126)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL94EVQVGIYGALN (SEQ ID NO: 127)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL95RPSEKIYGALN (SEQ ID NO: 128)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL96RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL97KTGDLIYGALN (SEQ ID NO: 130)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL98SCRVPIYGALN (SEQ ID NO: 131)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL99VASRGIYGALN (SEQ ID NO: 132)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL100RGRQNIYGALN (SEQ ID NO: 133)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL101AAPRGIYGALN (SEQ ID NO: 134)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL102SAPFKIYGALN (SEQ ID NO: 135)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL103LGMDDIYGALN (SEQ ID NO: 136)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL104NVRRGIYGALN (SEQ ID NO: 137)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL105NTSGRIYGALN (SEQ ID NO: 138)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL106LVSRPIYGALN (SEQ ID NO: 139)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL107WTNRPIYGALN (SEQ ID NO: 140)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL108RIPSAIYGALN (SEQ ID NO: 141)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL109GATRGIYGALN (SEQ ID NO: 142)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL110EGGSPIYGALN (SEQ ID NO: 143)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL111GASRGMFRALN (SEQ ID NO: 144)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL112GASGLVFSALN (SEQ ID NO: 145)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL113GASRGTHMALN (SEQ ID NO: 146)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL114GASSRFHNALN (SEQ ID NO: 147)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL115GASRTAFTALN (SEQ ID NO: 148)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL116GASRSTFSALN (SEQ ID NO: 149)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL117GASGPMFDALN (SEQ ID NO: 150)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL118GASHDLYGALN (SEQ ID NO: 151)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL119GASGTLFGALN (SEQ ID NO: 152)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL120GASKAAFGALN (SEQ ID NO: 153)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL121GASEGIVGALN (SEQ ID NO: 154)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL122GASHEIHVALN (SEQ ID NO: 155)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL123GASRGVFGALN (SEQ ID NO: 156)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL124GASGRVRGALN (SEQ ID NO: 157)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL125GASTGSFSALN (SEQ ID NO: 158)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL126GASGNSFDALN (SEQ ID NO: 159)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL127GASEQSYFALN (SEQ ID NO: 160)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL128GASFQFSALN (SEQ ID NO: 161)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL129GASAPRHSALN (SEQ ID NO: 162)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL130GASMPLFHALN (SEQ ID NO: 163)GATNLAD (SEQ ID NO: 2)QNVLSSPYT (SEQ ID NO: 3)AL131GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILSSPYT (SEQ ID NO: 252)AL132GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSMPYT (SEQ ID NO: 253)AL133GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLSEPET (SEQ ID NO: 254)AL134GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLYSPET (SEQ ID NO: 255)AL135GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLEEPYT (SEQ ID NO: 256)AL136GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLELPET (SEQ ID NO: 257)AL137GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLEMPET (SEQ ID NO: 258)AL138GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILESPET (SEQ ID NO: 259)AL139GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVIESPET (SEQ ID NO: 260)AL140GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVMESPET (SEQ ID NO: 261)AL141GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLESPET (SEQ ID NO: 262)AL142GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLYEPYT (SEQ ID NO: 263)AL143GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILSEPET (SEQ ID NO: 264)AL144GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVISEPET (SEQ ID NO: 265)AL145GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVMSEPET (SEQ ID NO: 266)AL146GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLSEPET (SEQ ID NO: 267)AL147GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QSVLFEPFS (SEQ ID NO: 268)AL148GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QSVLFEPFT (SEQ ID NO: 269)AL149GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILYSPET (SEQ ID NO: 270)AL150GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILSLPET (SEQ ID NO: 271)AL151GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNILSMPET (SEQ ID NO: 272)AL152GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLYMPET (SEQ ID NO: 273)AL153GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVISMPET (SEQ ID NO: 274)AL154GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVMSMPET (SEQ ID NO: 275)AL155GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLSMPET (SEQ ID NO: 276)AL156GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNIISSPET (SEQ ID NO: 277)AL157GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVLYLPET (SEQ ID NO: 278)AL158GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVIYSPET (SEQ ID NO: 279)AL159GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVMYSPET (SEQ ID NO: 280)AL160GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLYSPET (SEQ ID NO: 281)AL161GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVISLPET (SEQ ID NO: 282)AL162GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNVMSLPET (SEQ ID NO: 283)AL163GASENIYGALN (SEQ ID NO: 1)GATNLAD (SEQ ID NO: 2)QNLLSLPET (SEQ ID NO: 284)AL164RASAVIYGALN (SEQ ID NO: 129)GATGLAD (SEQ ID NO: 173)QNVLSSPYT (SEQ ID NO: 3)AL165GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLESPYT (SEQ ID NO: 177)AL166GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLSEPYT (SEQ ID NO: 182)AL167GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLSSPET (SEQ ID NO: 181)AL168GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLYSPYT (SEQ ID NO: 179)AL169GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNILSSPET (SEQ ID NO: 188)AL170GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNLLSSPET (SEQ ID NO: 206)AL171GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVISSPET (SEQ ID NO: 196)AL172GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVMSSPET (SEQ ID NO: 205)AL173GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLSLPET (SEQ ID NO: 185)AL174GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNVLSMPET (SEQ ID NO: 187)AL175GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QSVLFSPFS (SEQ ID NO: 207)AL176GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QNLLFQPLS (SEQ ID NO: 226)AL177GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QQVLFFPLL (SEQ ID NO: 213)AL178GASENIYGALN (SEQ ID NO: 1)GATGLAD (SEQ ID NO: 173)QSVLFSPFT (SEQ ID NO: 210)AL179RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLESPYT (SEQ ID NO: 177)AL180RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLSEPYT (SEQ ID NO: 182)AL181RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLSSPET (SEQ ID NO: 181)AL182RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLYSPYT (SEQ ID NO: 179)AL183RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNILSSPET (SEQ ID NO: 188)AL184RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNLLSSPET (SEQ ID NO: 206)AL185RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVISSPET (SEQ ID NO: 196)AL186RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVMSSPET (SEQ ID NO: 205)AL187RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLSLPET (SEQ ID NO: 185)AL188RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNVLSMPET (SEQ ID NO: 187)AL189RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QSVLFSPFS (SEQ ID NO: 207)AL190RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QNLLFQPLS (SEQ ID NO: 226)AL191RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QQVLFFPLL (SEQ ID NO: 213)AL192RASAVIYGALN (SEQ ID NO: 129)GATNLAD (SEQ ID NO: 2)QSVLFSPFT (SEQ ID NO: 210)AL193GASRSTFSALN (SEQ ID NO: 149)GATNLAD (SEQ ID NO: 2)QNVLSIPET (SEQ ID NO: 186)AL194GASMPLFHALN (SEQ ID NO: 163)GATNLAD (SEQ ID NO: 2)QNVLSIPET (SEQ ID NO: 186)AL195GASRSTFSALN (SEQ ID NO: 149)GATNLAD (SEQ ID NO: 2)QNVLEEPYT (SEQ ID NO: 256)AL196GASMPLFHALN (SEQ ID NO: 163)GATNLAD (SEQ ID NO: 2)QNVLEEPYT (SEQ ID NO: 256)

[0219]

[0220] Amino acid sequenceAH71EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYYINWVRQAPGKGLEWIGEIFPGWGNTHFSARFKGRATLSADKSKNTAYLQMNSLRAEDTAVYYCAGQDYGFLYWGQGTLVTVSS (SEQ ID NO: 285)AH72EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYYINWVRQAPGKGLEWIGEIFPGWGNTHFSRSPTPRATLSADKSKNTAYLQMNSLRAEDTAVYYCAGGDYGFLYWGQGTLVTVSS (SEQ ID NO: 286)AH73EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYYINWVRQAPGKGLEWIGEIFPGWGNSHVVSRFKGRATLSADKSKNTAYLQMNSLRAEDTAVYYCAGGDYGFLYWGQGTLVTVSS (SEQ ID NO: 287)AH85EVQLVESGGGLVQPGGSLRLSCAASGYTFSDYYINWVRQAPGKGLEWIGEIFPGWGNSHVVSRFKGRATLSADKSKNTAYLQMNSLRAEDTAVYYCAGQDYGFLYWGQGTLVTVSS (SEQ ID NO: 288)AL130DIQMTQSPSSLSASVGDRVTITCGASMPLFHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLSSPYTFGQGTKVEIK (SEQ ID NO: 289)AL135DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLEEPYTFGQGTKVEIK (SEQ ID NO: 290)AL165DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATGLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLESPYTFGQGTKVEIK (SEQ ID NO: 291)AL166DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATGLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLSEPYTFGQGTKVEIK (SEQ ID NO: 292)AL194DIQMTQSPSSLSASVGDRVTITCGASMPLFHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLSIPETFGQGTKVEIK (SEQ ID NO: 293)AL195DIQMTQSPSSLSASVGDRVTITCGASRSTFSALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLEEPYTFGQGTKVEIK (SEQ ID NO: 294)

[0221]

[0222] Tests for proliferation inhibitory activity on the U-87 MG cell line were performed in vitro using some of the affinity-optimized antibodies, and the results are shown in Table 9.

[0223]

[0224] AntibodyU-87 MG (GBM, HGF autocrine)Cell proliferation inhibition assay, IC50(nM)IC50FoldAffinity-optimized antibodyhuCM05huCM05 AH7111.395.58.5huCM05 AH7210.995.58.8huCM05 AH7310.995.58.8huCM05 AH8510.195.59.5huCM05 AL1305.045.09.0huCM05 AL1357.131.94.5huCM05 AL1656.839.05.7huCM05 AL1669.139.04.3huCM05 AL1949.694.59.8huCM05 AL19518.094.55.3

[0225]

[0226] As shown in Table 9, the IC50of the tumor cell proliferation inhibitory activity of the huCM05 affinity-optimized antibody in U-87 MG cells ranged from 5.0 to 18 nM, resulting in a 4.3- to 9.8-fold increase in efficacy compared to the parental huCM05 antibody. The above results were obtained by using some of the antibodies with the amino acid sequences presented in Table 6 to Table 8, but the affinity of the parent huCM05 antibody was optimized, and the selection of the antibodies was based on antigen affinity during the selection process, so the remaining affinity-optimized antibodies, and antibodies formed by combining the presented heavy and light chain variable region CDRs are also expected to have sufficiently equivalent effects.

[0227] For additional experiments, 10 types of affinity-optimized antibodies were prepared by combining the light and heavy chain variable regions. Specific light and heavy chain sequence combinations are shown in Table 10.

[0228]

[0229] Heavy chain variable regionLight chain variable regionhuCM05 AH71AH71 (SEQ ID NO: 285)huCM05-1 antibody light chain variable region(SEQ ID NO: 11)huCM05 AH85AH85 (SEQ ID NO: 288)huCM05-1 antibody light chain variable region(SEQ ID NO: 11)huCM05 AL194huCM05-1 antibody heavy chain variable region(SEQ ID NO: 12)AL194 (SEQ ID NO: 293)huCM05 A56AH85 (SEQ ID NO: 288)AL165 (SEQ ID NO: 291)huCM05 A65AH72 (SEQ ID NO: 285)AL130 (SEQ ID NO: 289)huCM05 A71AH73 (SEQ ID NO: 286)AL135 (SEQ ID NO: 290)huCM05 A72AH73 (SEQ ID NO: 287)AL165 (SEQ ID NO: 291)huCM05 A73AH73 (SEQ ID NO: 287)AL166 (SEQ ID NO: 292)huCM05 A76AH73 (SEQ ID NO: 287)AL195 (SEQ ID NO: 294)huCM05 A78AH71 (SEQ ID NO: 285)AL130 (SEQ ID NO: 289)

[0230]

[0231] Then, the tumor cell proliferation inhibitory activity assay was performed using the same method as in Example 1 above, and the results are shown in Table 11.

[0232]

[0233] AntibodyU-87 MG (GBM, HGF autocrine)Cell proliferation inhibition assay, IC50(nM)IC50FoldAffinity-optimized antibodyhuCM05huCM05 AH713.649.013.6huCM05 AH853.249.015.2huCM05 AL1945.349.09.2huCM05 A561.749.028.5huCM05 A621.849.027.6huCM05 A715.049.09.7huCM05 A723.649.013.8huCM05 A734.049.012.3huCM05 A764.349.011.3huCM05 A782.649.018.9

[0234]

[0235] As shown in Table 11, not only huCM05, but also 10 types of major antibodies formed by combining the light and heavy chain variable regions of the affinity-optimized antibody exhibited tumor cell proliferation inhibitory activity. In particular, it was confirmed that the 10 types of the antibodies have IC50of 1.7 to 5.3 nM, which were 9.2 to 28.5 times superior to the parent antibody huCM05 in inhibiting tumor cell proliferation.

[0236]

[0237] Example 5. Measurement of binding affinity to ECD (BIAcore)

[0238] Next, to measure the binding affinity of the c-MET antibody of the present disclosure to the extracellular domain (ECD), the binding of the c-MET antibody to human and cynomolgus monkey c-MET ECD was measured using BIAcore, for which human c-MET ECD (ACROBiosystems, MET-H5227) and cynomolgus monkey c-MET ECD (Sino Biological, 90304-C08H) were used.

[0239] First, an anti-Human IgG antibody (SouthernBiotech, 2047-01) specific for Fc was immobilized on the CM5 sensor chip at a level of 10000 RU to capture the anti-c-MET antibody. The antibody was diluted in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% (v / v) Surfactant P20) at a concentration of 1 - 2 μg / ml and injected into the CM5 chip immobilized with anti-Human Ig Fc at a flow rate of 30 μl / min for 10 to 120 seconds, followed by capture in the range of 150 - 200 RU. Sequential injections were performed, starting from the lower concentration, utilizing dilutions of 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM for each antigen. Binding was then achieved by injection for 5 min at a flow rate of 30 μl / min, followed by dissociation by injection of running buffer for 10 - 15 min. The chip was regenerated using 15 μl of 10 mM Glycine-HCl (pH 1.5). The binding and dissociation rates for each cycle were evaluated using the "1:1 Langmuir binding" model in BIAevaluation software version 4.1, and the BIAcore data are summarized in Table 12.

[0240]

[0241] Association constant(Kon, 1 / Ms)Dissociation constant(Koff, 1 / s)Affinity for antigen(KD, M)huCM05Human c-MET6.77 x 1052.148 x 10-43.173 x 10-10Cynomolgus monkey c-MET7.467 x 1053.447 x 10-44.616 x 10-10huCM05 AH71Human c-MET8.306 x 1058.301 x 10-59.993 x 10-11

[0242]

[0243] The above data demonstrates that the huCM05 antibody of the present disclosure binds to human and cynomolgus monkey c-MET ECD with excellent affinity.

[0244]

[0245] Example 6. Cross-reactivity of c-MET antibody to various receptors on cell surface

[0246] The specificity of the huCM05 antibody that binds specifically to c-MET according to the present disclosure and the cross-reactivity to other receptor tyrosine kinase antigens were analyzed by indirect ELISA method, wherein five antigens were selected for analysis among the major receptor tyrosine kinases: FGF R3, VEGFR R2, IGF IR, PDGF R, and RON.

[0247] In the present Example, human c-MET Fc chimera (R&D systems, 358-MT_CF), human FGF R3 (IIIc) Fc chimera (R&D systems, 766-FR), human IGF-I R (R&D systems, 391-GR-050), human PDGF Rβ Fc chimera (R&D systems, 385-PR_CF), human VEGF R2 Fc chimera (R&D systems, 357-KD_CF), and human MSP R / Ron (R&D systems, 1947-MS-050) were used as antigens.

[0248] Each antigen was diluted to a concentration of 1 μg / ml in 0.05 M carbonate-bicarbonate (Sigma, C3041) buffer and added to each well of a 96-well plate (Corning, #2592), which was then coated overnight at 4℃. The plate was washed once with TBS-T, and to inhibit non-specific binding, 200 μl of TBS-T containing 4% skim milk was added to each well and reacted at 37℃ for 1 hour. After washing 1 time with TBS-T buffer, the primary antibody was then sequentially diluted in TBS-T buffer containing 2% - skim milk from the highest concentration of 30 nM to 0.002 nM, and 100 μl of diluted antibody was added to each well and reacted at 37℃ for 2 hours. After washing three times with TBS-T buffer, 100 μl of anti-human kappa light chains-peroxidase (Sigma, A7164) diluted 1:5000 as a secondary antibody was added to each well, and reacted at 37℃ for 1 hour. After washing three times with TBS-T buffer, 100 μl of TMB solution (Sigma, T4444) was then added to each well to proceed with the color reaction, and then 50 μl of 2 N ammonium sulfate solution was added to each well to stop the reaction. Absorbance measurements were performed using a microplate reader, recording values at a wavelength of 450 nm, while utilizing a reference wavelength of 570 nm. The binding level of the anti-c-MET antibody to each antigen demonstrated a proportional relationship with the absorbance signal values, and the results are shown in Table 13.

[0249]

[0250] huCM05 binding (A450nm- A570nm)Ab. Concentration(nM)c-METIGF-IRRONPDGFRVEGFR2FGFR330.0002.552.510.000.000.000.000.010.010.000.010.010.026.0001.962.030.000.000.000.00-0.01-0.01-0.01-0.010.000.011.2001.811.740.000.000.000.00-0.01-0.01-0.01-0.010.000.010.2401.481.540.000.000.000.00-0.01-0.01-0.02-0.02-0.010.000.0480.760.760.000.000.000.00-0.01-0.01-0.02-0.010.000.000.0100.210.200.000.000.000.00-0.01-0.01-0.01-0.010.000.000.0020.050.050.000.000.000.00-0.01-0.01-0.01-0.010.000.00Blank0.000.000.000.000.000.000.000.000.000.000.000.00

[0251]

[0252] As shown in Table 13, it was confirmed that the huCM05 antibody of the present disclosure was specifically bound to c-MET, with little binding observed to the other antigens FGF R3, VEGFR R2, IGF IR, PDGF R and RON.

[0253]

[0254] Example 7. Preparation of c-MET-ADC (Antibody-Drug conjugate)

[0255] (1) Overview of preparation

[0256] ADCs containing the anti-c-MET antibody of the present disclosure were prepared using Synaffix's GlycoConnectTMtechnology. GlycoConnectTMtechnology involves enzymatic remodeling to introduce an azide group to the glycosylation site of an antibody, and preparing an antibody-drug conjugate (ADC) through a simple click reaction (see FIG. 2). Since one glycosylation site is present on each heavy chain of an antibody, the technology has the advantage of uniform preparation of ADC with desired drug-antibody ratios (DARs).

[0257] ADC was prepared by reacting huCM05, the anti-c-MET antibody of the present disclosure, with a linker-payload conjugate containing MMAE (Formula 9), and named CM05-Auri.

[0258] [Formula 9]

[0259]

[0260]

[0261] The ADC of the present disclosure is prepared through two steps: enzymatic remodeling of the antibody and drug attachment. The preparation process of CM05-Auri (CM05-BCN-HS-(vc-PABC-MMAE)2) is shown in FIG. 3. Briefly, it may be prepared by first adding and reacting endoglycosidase, glycosyltransferase (GalNAc-transferase), UDP-sugar, and alkaline phosphate with the antibody for antibody remodeling, and then mixing the remodeled antibody with a linker-payload conjugate, followed by conjugation via a metal-free click chemistry reaction.

[0262]

[0263] (2) Glycan remodeling

[0264] The huCM05 antibody was added to TBS (pH 7.5) at a concentration of 25 mg / ml, then endoglycosidase, glycosyltransferase, UDP-sugar, and MnCl2were added and incubated at 30℃ overnight. To remove glycan remodeling components, the resulting product was purified with Protein A and added to PBS (pH 7.4) to a concentration of 25 mg / ml. After overnight incubation, samples were digested with IdeS and then subjected to MS analysis (FIG. 4). The results showed that the huCM05 antibody was remodeled into an azide group-modified form (azido-huCM05).

[0265]

[0266] (3) Conjugation with linker-payload

[0267] The azido-huCM05 antibody (15 mg / ml, PBS pH 7.4) and a linker-payload conjugate containing MMAE (BCN-HS-(vc-PABC-MMAE)2) (7 equivalents, i.e., 3.5 equivalents per azide) were reacted under 10% DMF as a co-solvent, and incubated overnight at room temperature.

[0268] The samples cleaved by IdeS were then verified by MS analysis (FIG. 5), and it was confirmed that the desired ADCs for each payload were well prepared (FIG. 6).

[0269] As Comparative Examples, Teliso-V (telisotuzumab vedotin) was prepared with reference to International Publication No. 2017 / 201204, and B12-MMAE (BCN-HS-(va-PABC-MMAE)2conjugated to B12 antibody, an anti-gp120 antibody) was prepared in a similar manner as described above.

[0270]

[0271] (5) ADC Characterization

[0272] The results of analyzing the characteristics of the ADCs of Example of the present disclosure and Comparative Example are shown in Table 14.

[0273]

[0274] CM05-AuriB12-MMAEFormulationhistidine 20 mM, sucrose 6 %, tween 20 0.04 %, pH 6.0histidine 20 mM, sucrose 6 %, tween 20 0.04 %, pH 6.0Mass Spectrometry27124 Da(Fc / 2-fragment)27121 Da(Fc / 2-fragment)Free payload level(RP-HPLC / UPLC-MS)1 %1 %Non-reducing CE-SDS / SDS-PAGE≥95 % main intact antibody band≥ 95 % main intact antibody bandMonomer content(SEC-HPLC)98.9 %≥99 %Average DAR (RP-UPLC) DTT-treated3.93.65Endotoxin levels(LAL assay)< 0.1 EU / mg< 0.25 EU / mg

[0275]

[0276] Example 8. Antigen binding assay of c-MET-ADC

[0277] (1) Target binding evaluation using Surface Plasmon Resonance (SPR)

[0278] The binding affinity of CM05-Auri and naked antibody (huCM05) was compared using SPR instrument (BIAcore T200). An anti-human Fc antibody (SouthernBiotech) was immobilized on a CM5 sensor chip and antibody or ADC was captured on the immobilized surface. The binding and dissociation times were then measured using the target antigen protein.

[0279] As a result, the KDvalues of huCM05 and CM05-Auri for c-MET were found to be 462 pM and 442 pM, respectively (Table 15). This shows that the conversion of huCM05 to CM05-Auri does not change its binding ability to the target protein.

[0280]

[0281] AntibodykakdKDhuCM054.03 x 1051.86 x 10-4462 pMCM05-Auri4.28 x 1051.89 x 10-4442 pM

[0282]

[0283] (2) Evaluation of target protein binding affinity using enzyme-linked immunosorbent assay (ELISA)

[0284] The binding activity of CM05-Auri and Teliso-V to the target protein c-MET was compared using an ELISA assay. CM05-Auri or Teliso-V was added to a plate coated with recombinant c-MET protein to assess binding levels.

[0285] The results showed that CM05-Auri had a target binding EC50of 0.2 nM, which was superior to Teliso-V having a target binding EC50of 0.65 nM (FIG. 7).

[0286]

[0287] (3) Evaluation of binding affinity to target protein-expressing cells using FACS (Fluorescence-activated Cell Sorting)

[0288] Binding affinity was analyzed using NCI-H441 and NCI-H1975 cell lines expressing high and medium levels of c-MET, respectively. After incubating the cells with CM05-Auri or Teliso-V, fluorescent secondary antibodies were attached and ADC bound to the cells was measured using FACS analysis.

[0289] The results showed that in the NCI-H441 cell line, the binding EC50of Teliso-V was 0.67 nM, while the binding EC50of CM05-Auri was 0.15 nM, indicating a better binding affinity than Teliso-V. In experiments using the NCI-H1975 cell line, the binding EC50of CM05-Auri was 0.32 nM, showing a better binding affinity than Teliso-V at 1.43 nM (FIG. 8).

[0290]

[0291] Example 9. Internalization analysis of c-MET-ADC

[0292] (1) Evaluation of internalization rate

[0293] To determine whether the internalization rate of the huCM05 antibody remained unchanged when converted to ADC, a comparative analysis was conducted between the internalization rates of the naked antibody (huCM05) and CM05-Auri. After incubating ADC with HT-29 cells expressing the target protein, unbound ADC was removed and internalization was induced at 37℃. The amount of ADC that entered the cells was then measured by FACS at each time point (30-minute intervals, up to 2 hours).

[0294] The results showed that the internalization rate of ADC was similar to that of the huCM05 antibody, confirming that the conversion of huCM05 antibody to CM05-Auri did not change the internalization rate (FIG. 9).

[0295]

[0296] (2) Evaluation of continuous internalization rate

[0297] To measure the amount of ADC entering the cells due to internalization, a continuous internalization assay was performed. H441 and H1975 cell lines were treated with ADC and incubated at 37℃, and the amount of ADC entering the cells was measured by FACS over time.

[0298] The results showed that CM05-Auri entered the cells more than Teliso-V in both cell lines tested. After 6 hours of treatment in H441 and H1975 cell lines, CM05-Auri had 1.6- and 1.9-times higher levels than Teliso-V, respectively (Table 16 and FIG. 10).

[0299]

[0300] Cell lineADCTimeMFI (1st)MFI (2nd)MFI (3rd)H441CM05-Auri000024486437436306496546943818Teliso-V000022952222424236353423492552H1975CM05-Auri00002132254775761538745999Teliso-V000028212505276801335610

[0301]

[0302] Since ADC mainly exhibits cytotoxic activity after internalization, these experimental results suggest that CM05-Auri has better efficacy than Teliso-V.

[0303]

[0304] Example 10. Evaluation of in vitro efficacy of c-MET-ADC

[0305] (1) In vitro efficacy evaluation using cancer cell lines

[0306] First, 100 μl of cells were seeded at the indicated density in 96-well plate. The plate was incubated in a humidified incubator at 37℃ under 5% CO2for 24 hours. Next, serial dilutions of CM05-Auri, Teliso-V, or MMAE free drug were prepared (test agents) using culture medium containing 10% FBS. After removing the culture medium from the 96-well plate, 200 μl of the prepared test agents were added to the plate containing cells, respectively. The plate was then incubated in a humidified incubator at 37℃ under 5% CO2for 6 days. After removing the culture medium, 100 μl of 10% TCA fixation solution was added to each well and the plate was incubated at 4℃ for at least 2 hours. The plate was washed three times by immersing it in a tub filled with distilled water, and then the plate was placed on a paper towel and gently tapped to remove excess water. After air drying, 80 μl of SRB solution (0.4% SRB in 1% acetic acid) was added to each well and stained for 30 minutes at room temperature. The plate was washed three times by immersion in a tub filled with 0.1% acetic acid and air dried at room temperature. Then, 150 μl of 10 mM Trizma base solution was added to each well and the plate was left at room temperature for 30 minutes to allow the protein-bound dye to dissolve, and the absorbance was measured at 540 nm using a microplate reader.

[0307] Cell growth was calculated as follows.

[0308]

[0309] The EC50and maximum killing values (percentage of cell growth inhibition at 1 μg / ml ADC) were calculated by constructing a dose-response curve between concentration and percentage of cell growth, employing the curve-fitting method with statistical analysis using Prism software. The approximate number of c-MET molecules on the cell surface was determined by FACS analysis as antibody-binding capacity for c-MET antibody using QIFIKIT (Agilent).

[0310] The results showed that the EC50of CM05-Auri was all in the subnanomolar level in cell lines with c-MET expression levels of 29,000 or more, and had a lower EC50than Teliso-V, indicating that CM05-Auri has more powerful efficacy than Teliso-V in vitro (Table 17). On the other hand, two types of ADCs both showed no efficacy in cell lines that do not express c-MET, indicating that the efficacy of the ADCs is dependent on the target protein.

[0311]

[0312] Cell lineMutationc-Met expressionMMAE EC50CM05-AuriEC50(nM)Teliso-VEC50(nM)H2444KRAS G12V138,1930.5490.0150.285H1373KRAS G12C86,3010.8900.649N / DH441KRAS G12V72,2910.3000.0440.157HCC827EGFR Ex19del59,541-0.0674.298H1573KRAS G12A55,810-0.09710.440SW900KRAS G12V50,5080.2800.034>100H358KRAS G12C37,2990.5170.0351.292HT-29BRAF V600E, PIK3CA P449T35,3980.3300.0410.143H1975T790M, L858R31,8420.3630.1393.873Ls411N-29,0320.3270.0430.164H520c-MET negative3330.864>100>100H661c-MET negative1520.665>100>100

[0313]

[0314] (2) Evaluation of bystander effect

[0315] The bystander effect of CM05-Auri was tested using H441, a c-MET overexpressing cell line, and H520 and H661, cell lines not expressing c-MET. When the medium that had been treated with CM05-Auri to H441 cells for 5 days was then treated with H520 and H661 cells for 5 days, cell growth was also inhibited in c-MET negative cell lines (FIG. 11). These results show that CM05-Auri releases drug to cells expressing the target protein, and the released drug kills cells not expressing the target protein, suggesting that CM05-Auri has the bystander effect.

[0316]

[0317] Example 11. Evaluation of in vivo efficacy of c-MET-ADC

[0318] (1) Study of in vivo activity of CM05-Auri in H441 CDX model

[0319] To evaluate the antitumor effects of CM05-Auri and huCM05 in the H441 NSCLC xenograft model, BALB / c-nude mice were inoculated with H441 cells to form tumors. When the tumor size reached approximately 200 mm3, the mice were grouped and injected with the drugs into the intraperitoneal space once a week for a total of four times.

[0320] On Day 35, the tumor size in the CM05-Auri 1 mg / kg administration group was 62.45 mm3, which showed a significant inhibition of tumor growth when compared to the vehicle control (760.08 mm3) and huCM05 3 mg / kg administration group (692.62 mm3) (FIG. 12).

[0321]

[0322] (2) Study of in vivo activity of CM05-Auri in H1975 CDX model

[0323] To evaluate the antitumor effects of CM05-Auri and huCM05 in the H1975 NSCLC xenograft model, BALB / c-nude mice were inoculated with H1975 cells to form tumors. When the tumor size reached approximately 200 mm3, the mice were grouped and injected with the drugs into the intraperitoneal space once a week for a total of four times.

[0324] On Day 35, the tumor size in the CM05-Auri 3 mg / kg administration group was 167.71 mm3, which showed a significant inhibition of tumor growth when compared to the vehicle control (2318.13 mm3) and CM05 3 mg / kg administration group (2496.14 mm3) (FIG. 13).

[0325]

[0326] (3) Study of in vivo activity of CM05-Auri in HT-29 CDX model

[0327] To evaluate the antitumor effects of CM05-Auri and huCM05 in the HT-29 CRC xenograft model, BALB / c-nude mice were inoculated with HT-29 cells to form tumors. When the tumor size reached approximately 110 mm3, the mice were grouped and injected with the drugs into the intraperitoneal space once a week for a total of four times.

[0328] On Day 34, the tumor size in the CM05-Auri 3 mg / kg administration group was 175.72 mm3, which showed a significant inhibition of tumor growth when compared to the vehicle control (2315.64 mm3) and CM05 3 mg / kg administration group (2411.92 mm3) (FIG. 14).

[0329]

[0330] (4) Comparative study of in vivo activity of CM05-Auri and Teliso-V in HCC827 CDX model

[0331] To evaluate the antitumor effects of CM05-Auri compared to Teliso-V in the HCC827 NSCLC xenograft model, BALB / c-nude mice were inoculated with HCC827 cells to form tumors. When the tumor size reached approximately 200 mm3, mice were grouped and each drug was injected once into the intraperitoneal space.

[0332] On Day 24, the tumor size in the CM05-Auri 4.5 mg / kg administration group was 261.74 mm3, which was significantly reduced compared to the vehicle control group (P < 0.05). On the other hand, the tumor size in the Teliso-V 4.5 mg / kg administration group was 836.84 mm3, showing no significant difference compared to the vehicle control group (FIG. 15).

[0333]

[0334] (5) Comparative study of in vivo activity of CM05-Auri and Teliso-V in H1573 CDX model

[0335] To evaluate the antitumor effects of CM05-Auri compared to Teliso-V in the H1573 NSCLC xenograft model, NOD / SCID mice were inoculated with H1573 cells to form tumors. When the tumor size reached approximately 300 mm3, mice were grouped and each drug was injected once into the intraperitoneal space.

[0336] On Day 35, the tumor size in the CM05-Auri 9 mg / kg administration group was 475.39 mm3, which was significantly reduced compared to the vehicle control group (1859.50 mm3, P < 0.05) and the Teliso-V 9 mg / kg administration group (954.74 mm3, P < 0.05) (FIG. 16).

[0337]

[0338] (6) Comparative study of in vivo activity of CM05-Auri and Teliso-V in LU11681 PDX model

[0339] To evaluate the antitumor effects of CM05-Auri compared to Teliso-V in the LU11681 NSCLC xenograft model, NOD / SCID mice were inoculated subcutaneously with tumor fragments (2 - 3 mm in diameter) on the right flank to form tumors. When the tumor size reached approximately 300 mm3, mice were grouped and each drug was injected once into the intraperitoneal space.

[0340] On Day 25, the tumor sizes in the CM05-Auri 4.5 mg / kg and 9 mg / kg administration groups were 1050.75 mm3and 590.44 mm3, respectively, which were significantly reduced compared to the Teliso-V 4.5 mg / kg and 9 mg / kg administration groups (1932.13 mm3and 1428.05 mm3, respectively, P < 0.05) (FIG. 17).

[0341]

[0342] (7) Comparative study of in vivo activity of CM05-Auri and Teliso-V in LU5165 PDX model

[0343] To evaluate the antitumor effects of CM05-Auri compared to Teliso-V in the LU5165 NSCLC xenograft model, NOD / SCID mice were inoculated subcutaneously with tumor fragments (2 - 3 mm in diameter) on the right flank to form tumors. When the tumor size reached approximately 300 mm3, mice were grouped and each drug was injected once into the intraperitoneal space.

[0344] On Day 35, the tumor sizes in the CM05-Auri 4.5 mg / kg and 9 mg / kg administration groups were 772.50 mm3and 477.92 mm3, respectively, which were significantly reduced compared to the Teliso-V 4.5 mg / kg and 9 mg / kg administration groups (1103.78 mm3and 1002.88 mm3, respectively) (FIG. 18).

[0345]

[0346] Example 12. Blood stability test of c-MET-ADC

[0347] To evaluate the stability of CM05-Auri, the blood stability of CM05-Auri was tested using cynomolgus monkeys. As a result, CM05-Auri was found to be highly stable in cynomolgus monkeys (FIG. 19). These results support that CM05-Auri is likely to be stable in humans.

[0348]

[0349] From the above description, those skilled in the art to which the present disclosure pertains will understand that the present disclosure may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure is indicated by the following claims rather than the detailed description, and should be construed as including all changes or modifications derived from the meaning and scope of the claims and equivalent concepts within the scope of the present disclosure.

Claims

1.An immunoconjugate represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:[Formula 1]in Formula 1 above,Ab is an antibody comprising a light chain variable region comprising light chain CDR1 set forth in SEQ ID NO: 1; light chain CDR2 set forth in SEQ ID NO: 2; and light chain CDR3 set forth in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 set forth in SEQ ID NO: 4, heavy chain CDR2 set forth in SEQ ID NO: 5; and heavy chain CDR3 set forth in SEQ ID NO: 6, an affinity-optimized antibody thereof, or an antigen-binding fragment thereof;the Ab comprises a terminal GlcNAc moiety of the following Formula 2,[Formula 2]wherein S is GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) as a sugar moiety,Fuc is fucose, anda is 0 or 1;a terminal azide group of the terminal GlcNAc moiety of the Ab binds to the BCN (Bicyclo[6.1.0]non-4-yne) ring to form a triazole moiety of the following Formula 3,[Formula 3]wherein * is linked to the S in the antibody and ** is linked to the SU; andSUis a group connecting the BCN ring and LUand is represented by the following Formula 4,[Formula 4]wherein * is linked to the BCN ring, and ** is linked to the LU,RAand RBare each independently -H or -C1-3alkyl,n is an integer from 1 to 5;LUis a single bond or a branched linker, andwhen LUis a branched linker, LUis represented by the following Formula 5,[Formula 5]wherein * is linked to the SU, and ** is linked to the CL,M1and M2are each independently -C(=O)- or -OC(=O)-,q1and q2are each independently integers from 2 to 4; andCLis a cleavable linker represented by the following Formula 6,[Formula 6]wherein Dipeptide is -Val-Cit-, -Val-Ala-, -Phe-Lys- or -Glu-Ala-,Rx is each independently selected from -C1-3alkyl, -halo, or -OH,r is an integer from 0 to 4;D is a cytotoxic drug moiety; andp is an integer from 1 to 3.2.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the Ab comprises a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8.3.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the Ab comprises a hinge region set forth in any one of SEQ ID NO: 19 to SEQ ID NO: 26.4.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the affinity-optimized antibody comprises at least one amino acid sequence substitution in an antibody comprising a light chain variable region comprising light chain CDR1 set forth in SEQ ID NO: 1; light chain CDR2 set forth in SEQ ID NO: 2; and light chain CDR3 set forth in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 set forth in SEQ ID NO: 4; heavy chain CDR2 set forth in SEQ ID NO: 5; and heavy chain CDR3 set forth in SEQ ID NO: 6, wherein(i) in the light chain CDR1, G at position 1 is substituted with A, E, K, L, N, R, S, V or W; A at position 2 is substituted with C, G, I, P, S, T or V; S at position 3 is substituted with G, M, N, P, Q, R, S or T; E at position 4 is substituted with A, D, F, G, H, K, M, Q, R, S, T or V; N at position 5 is substituted with A, D, E, G, K, L, P, Q, R, S, T or V; I at position 6 is substituted with A, F, L, M, Q, R, S, T or V; Y at position 7 is substituted with F, H, R or V; or G at position 8 is substituted with D, F, H, M, N, R, S, T or V;(ii) in the light chain CDR2, G at position 1 is substituted with D, F, H, K, P, Q, S, V or Y; T at position 3 is substituted with Q; or N at position 4 is substituted with G;(iii) in the light chain CDR3, Q at position 1 is substituted with E, G, I, M or N; N at position 2 is substituted with A, D, E, H, L, Q, S or T; V at position 3 is substituted with I, L, M, N, Q, S or T; L at position 4 is substituted with F, H, I, M, R, S, V, W or Y; S at position 5 is substituted with C, D, E, F, G, H, K, L, N, Q, R, T, V or Y; S at position 6 is substituted with D, E, F, G, H, I, L, M, N, P, Q, R, T, V or Y; P at position 7 is substituted with A, D, E, G, N, Q, S or V; Y at position 8 is substituted with E, F, L, M or Q; or T at position 9 is substituted with D, F, G, I, L, N, S, V, W or Y;(iv) in the heavy chain CDR1, D at position 1 is substituted with G or Q; Y at position 2 is substituted with Q; or I at position 4 is substituted with A or Q;(v) in the heavy chain CDR2, F at position 3 is substituted with D, E, W or Y; G at position 5 is substituted with D, H or Y; S at position 6 is substituted with F, P, W or Y; G at position 7 is substituted with A, F, L, N or T; N at position 8 is substituted with F, P, S, T or Y; T at position 9 is substituted with A, D, E, F, G, H, L, P, S or V; H at position 10 is A, D, F, M, R, S, T, V, W or Y; F at position 11 is substituted with G, H, I, L, M, N, P, Q, V or Y; S at position 12 is substituted with A, D, G, H, I, L, P, T or V; A at position 13 is substituted with D, E, F, G, H, I, K, L, M, P, R, S, T, V or Y; R at position 14 is substituted with A, E, G, H, L, N, P, Q, S, W or Y; F at position 15 is substituted with D, E, G, L, M, P, R, S, V or W; K at position 16 is substituted with A, E, F, G, H, L, R, S, T, V or Y; or G at position 17 is substituted with E, F, H, L, M, N, P, Q, R, S, T, V or W; or(vi) in the heavy chain CDR3, G at position 1 is substituted with E, F, H, N, Q, V or W; D at position 2 is substituted with E; Y at position 3 is substituted with L, Q, T or V; G at position 4 is W; F at position 5 is substituted with L or Y; L at position 6 is substituted with Q, S or Y; or Y at position 7 is substituted with C, L, M, N or Q,wherein the light chain CDR1 comprises 0 to 5 substitutions, the light chain CDR2 comprises 0 to 1 substitution, the light chain CDR3 comprises 0 to 7 substitutions, the heavy chain CDR1 comprises 0 to 1 substitution, the heavy chain CDR2 comprises 0 to 11 substitutions, and the heavy chain CDR3 comprises 0 to 6 substitutions.5.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the affinity-optimized antibody comprises a light chain variable region comprising light chain CDR1 set forth in any one of SEQ ID NO: 1, and SEQ ID NO: 124 to SEQ ID NO: 163; light chain CDR2 set forth in any one of SEQ ID NO: 2, and SEQ ID NO: 164 to SEQ ID NO: 174; and light chain CDR3 set forth in any one of SEQ ID NO: 3, and SEQ ID NO: 175 to SEQ ID NO: 284, anda heavy chain variable region comprising heavy chain CDR1 set forth in any one of SEQ ID NO: 4, and SEQ ID NO: 36 to SEQ ID NO: 40; heavy chain CDR2 set forth in any one of SEQ ID NO: 5, and SEQ ID NO: 41 to SEQ ID NO: 110; and heavy chain CDR3 set forth in any one of SEQ ID NO: 6, and SEQ ID NO: 111 to SEQ ID NO: 123.6.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the affinity-optimized antibody comprises a light chain variable region set forth in any one of SEQ ID NO: 11, and SEQ ID NO: 289 to SEQ ID NO: 294, and a heavy chain variable region set forth in any one of SEQ ID NO: 12, and SEQ ID NO: 285 to SEQ ID NO: 288.7.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the affinity-optimized antibody comprises(a) a light chain variable region set forth in SEQ ID NO: 11 and a heavy chain variable region set forth in SEQ ID NO: 285;(b) a light chain variable region set forth in SEQ ID NO: 11 and a heavy chain variable region set forth in SEQ ID NO: 288;(c) a light chain variable region set forth in SEQ ID NO: 293 and a heavy chain variable region set forth in SEQ ID NO: 12;(d) a light chain variable region set forth in SEQ ID NO: 291 and a heavy chain variable region set forth in SEQ ID NO: 288;(e) a light chain variable region set forth in SEQ ID NO: 289 and a heavy chain variable region set forth in SEQ ID NO: 285;(f) a light chain variable region set forth in SEQ ID NO: 290 and a heavy chain variable region set forth in SEQ ID NO: 286;(g) a light chain variable region set forth in SEQ ID NO: 291 and a heavy chain variable region set forth in SEQ ID NO: 287;(h) a light chain variable region set forth in SEQ ID NO: 292 and a heavy chain variable region set forth in SEQ ID NO: 287;(i) a light chain variable region set forth in SEQ ID NO: 294 and a heavy chain variable region set forth in SEQ ID NO: 287; and(j) a light chain variable region set forth in SEQ ID NO: 289 and a heavy chain variable region set forth in SEQ ID NO: 285.8.The immunoconjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof of claim 1, wherein the cytotoxic drug moiety is a monomethyl auristatin E (MMAE), which is an auristatin compound.9.A pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate of any one of claims 1 to 8, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient.10.The pharmaceutical composition of claim 9, wherein the cancer is a c-MET positive cancer.

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