Novel immune complexes targeting c-MET and their applications

Abstract

The present invention relates to an immune complex targeting c-MET, and more specifically, to an immune complex comprising an anti-c-MET antibody having a specific CDR sequence including a terminal GlcNAc moiety, or its antigen-binding fragment, a linker, and a cytotoxic drug moiety, and to a pharmaceutical composition for the prevention or treatment of cancer containing the immune complex as an active ingredient. The c-MET-targeting immune complex of the present invention kills cancer cells at a high level in vivo and exhibits excellent in vivo stability compared to other c-MET-targeted ADCs, and can therefore be used very effectively for cancer prevention or treatment.

Description

[Technical Field]

[0001] The present invention relates to an immune complex targeting c-MET, and more specifically to an immune complex comprising an anti-c-MET antibody having a specific CDR sequence including a terminal GlcNAc moiety, an antigen-binding fragment thereof, a linker, and a cytotoxic drug moiety, as well as a pharmaceutical composition for the prevention or treatment of cancer comprising the immune complex as an active ingredient. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are one of the fastest-growing types of anticancer drugs in recent years. ADCs consist of a cytotoxic drug (payload), an antibody (Ab), and a linker that connects them, and are designed to be specifically delivered to cells expressing the target antigen of the Ab, thereby broadening the therapeutic index of the drug (Joshua Z. Drago et al., Nature Reviews Clinical Oncology, 18(6), 327).

[0003] On the other hand, c-MET is a protein encoded by the human MET gene, also known as the hepatocyte growth factor receptor (HGFR), and is a single-pass plasma membrane protein with tyrosine kinase activity in its intracellular domain. c-MET is mainly expressed in epithelial cells and regulates cell proliferation and wound healing. Hepatocyte growth factor binds to cMET as a ligand, activating the dimeric c-MET. Aberrant regulation, such as overexpression or abnormal activation of c-MET, is associated with cancer development and can worsen cancer prognosis. c-MET overexpression is known in various cancer types, including NSCLC and colorectal cancer.

[0004] AbbVie's c-MET-targeted ADC, telisotuzumab vedotin (Teliso-V), showed high efficacy in NSCLC overexpressing c-MET, demonstrating the potential of c-MET-targeted ADCs (Camidge, DRet al., J.Clin.Oncol. 2022, 40:9016). However, to date, no c-MET-targeted ADCs have received FDA approval in the United States, and there remains a strong demand for the development of new c-MET-targeted ADCs with superior anti-cancer efficacy. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The inventors of this invention have completed the present invention by developing a novel c-MET antibody, conjugating it to a drug-linker complex via enzymatic remodeling to produce a novel c-MET targeted immune complex, and confirming the excellent anticancer effect of the produced immune complex. [Means for solving the problem]

[0006] One object of the present invention is to provide an immune complex comprising an anti-c-MET antibody or its antigen-binding fragment, a linker, and a cytotoxic drug moiety, its stereoisomer, or pharmaceutically acceptable salts thereof.

[0007] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the immune complex, its stereoisomers, or pharmaceutically acceptable salts thereof. [Effects of the Invention]

[0008] The c-MET-targeting immune complex of the present invention can kill cancer cells at a high level in vivo and exhibits superior in vivo stability compared to other c-MET-targeted ADCs, making it extremely useful for cancer prevention or treatment. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram of the phagemid vector used to produce an affinity-optimized antibody for huCM05. [Figure 2] This diagram schematically illustrates the enzymatic remodeling technique used in the production of the immune complex of the present invention. [Figure 3] This document illustrates the process for producing the CM05-Auri immune complex of the present invention. [Figure 4] This shows the MS analysis results that confirmed azido-huCM05. [Figure 5] This shows the MS analysis results that confirmed CM05-Auri. [Figure 6] This shows the structure of CM05-Auri. [Figure 7] This shows the results of target protein binding affinity evaluation using ELISA. [Figure 8] This shows the results of evaluating the binding affinity of target proteins to cells expressing those proteins using FACS. [Figure 9] This shows the results of a comparative evaluation of the internalization rates of huCM05 antibody and CM05-Auri. [Figure 10] This shows the results of serial internalization analysis performed on huCM05 antibody and CM05-Auri. [Figure 11] This shows the results of confirming the bystander effect of CM05-Auri. [Figure 12] This shows the results of confirming the in vivo activity of CM05-Auri in the H441CDX model. [Figure 13] This shows the results of confirming the in vivo activity of CM05-Auri in the H1975CDX model. [Figure 14] This shows the results of confirming the in vivo activity of CM05-Auri in the HT-29CDX model. [Figure 15] This shows the results of confirming the in vivo activity of CM05-Auri in the HCC827CDX model. [Figure 16] This shows the results of confirming the in vivo activity of CM05-Auri in the H1573CDX model. [Figure 17] This shows the results of confirming the in vivo activity of CM05-Auri in the LU11681PDX model. [Figure 18] This shows the results of confirming the in vivo activity of CM05-Auri in the LU5165PDX model. [Figure 19] This shows the results of confirming the circulatory stability of CM05-Auri in cynomolgus monkeys. [Modes for carrying out the invention]

[0010] This can be explained in detail as follows. On the other hand, each description and embodiment disclosed in this invention can also be applied to each other description and embodiment. That is, any combination of the various components disclosed in this invention falls within the scope of this invention. Furthermore, the scope of this invention cannot be considered to be limited by the specific descriptions described below.

[0011] I. immunoconjugate One aspect of the present invention for achieving the above objective is an immunocomplex represented by the following formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [ka]

[0012] In the above formula 1, Ab is an antibody comprising a light chain variable region including light chain CDR1 described in SEQ ID NO: 1; light chain CDR2 described in SEQ ID NO: 2; and light chain CDR3 described in SEQ ID NO: 3, and a heavy chain variable region comprising heavy chain CDR1 described in SEQ ID NO: 4; heavy chain CDR2 described in SEQ ID NO: 5; and heavy chain CDR3 described in SEQ ID NO: 6, an affinity-optimized antibody thereof, or an antigen-binding fragment thereof; The aforementioned Ab includes the terminal GlcNAc portion of the following formula 2, [Chemical formula] (Here, S is GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) as the sugar moiety, Fuc is fucose, a is 0 or 1); The terminal azide group of the terminal GlcNAc moiety in Ab binds to the BCN (Bicyclo[6.1.0]nonyne) ring to form the triazole moiety of the following formula 3, [Chemical formula] (Here, * is linked to S of the antibody, and ** is linked to S ); S U is represented by the following formula 4 as a group linking the BCN ring and L U , [Chemical formula] (Here, * is linked to the BCN ring, and ** is linked to L U , R A and R B are each independently -H or -C 1-3 alkyl, n is an integer from 1 to 5); L U is a single bond or a branched linker, L U When L U is a branched linker, L [Chemical formula]

[0013] (Here, * is linked to S U , and ** is linked to C L , M1 and M2 are independently -C(=O)- or -OC(=O)-, q1 and q2 are each independent integers between 2 and 4. C L As a severable linker, it is represented by the following equation 6: [ka] (Here, Dipeptide is -Val-Cit-, -Val-Ala-, -Phe-Lys- or -Glu-Ala-, Each Rx is independently -C 1-3 Selected from alkyl, -halo, or -OH, r is an integer between 0 and 4. D is the cytotoxic drug portion; and p is an integer between 1 and 3.

[0014] In this invention, the term "immune complex" refers to a complex in which a drug-linker complex is linked to an antibody or its antigen-binding fragment, and specifically has the structure of Formula 1 below. [ka] In this invention, the term "drug-linker complex" means that the antibody or its antigen-binding fragment is not linked to the substance for the production of an immune complex, and can be bound to any antibody or its antigen-binding fragment as needed and used as an immune complex.

[0015] When administered in vivo, the aforementioned immune complex allows the antibody or its antigen-binding fragment to bind to the target antigen, and subsequently release the drug, thereby enabling the drug to act on target cells and / or surrounding cells. This is expected to result in superior efficacy and reduced side effects as a targeted drug.

[0016] In this invention, the term "antibody" refers to a protein molecule that acts as a ligand that specifically recognizes an antigen, including immunoglobulin molecules that are immunologically reactive with a particular antigen, and includes polyclonal antibodies, monoclonal antibodies, and whole antibodies. The term also includes chimeric antibodies and bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of the antibody constant region, and artificial antibodies based on protein scaffolds that possess the ability to bind to FcRn. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes subtypes such as IgG1, IgG2, IgG3, and IgG4.

[0017] In this invention, the terms "fragment," "antibody fragment," and "antigen-binding fragment" refer to any fragment of the antibody of this invention that possesses the antigen-binding function of the antibody, and are used interchangeably. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, dsFv, and scFv.

[0018] The aforementioned Fab has a structure with variable regions of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1 domain) of the heavy chain, and possesses one antigen-binding site. An antigen-binding fragment or antibody fragment of an antibody molecule refers to a fragment that possesses antigen-binding function. 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. The F(ab')2 antibody is produced with the cysteine ​​residues in the hinge region of Fab' forming disulfide bonds. Fd refers to the heavy chain portion included in the Fab fragment. Fv (variable fragment) refers to the minimal antibody fragment that has only the heavy chain variable region and the light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, while in single chain Fv (scFv), the heavy chain variable region and the short chain variable region are generally linked by covalent bonds via a peptide linker, or directly linked at the C-terminus, and can form a dimer-like structure. Although not limited to these, such antibody fragments can be obtained using proteolytic enzymes (for example, enzymatic digestion of the whole antibody with papain yields Fab, and digestion with pepsin yields the F(ab')2 fragment), or they can be produced by genetic engineering techniques.

[0019] c-MET (Hepatocyte growth factor receptor) is a type of RTK and is the 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)). Abnormal activation of c-MET by HGF is known to be one of the representative mechanisms of tumorigenesis and is associated with tumor growth, inhibition of apoptosis, angiogenesis, invasion, and metastasis (Bottaro DP et al., Science 251:802-804 (1991), Day RM et al., Oncogene 18:3399-3406 (1999)). Furthermore, abnormal activation of c-MET due to mutations and amplification has been reported to be associated with a variety of cancers, including lung cancer, colorectal cancer, head and neck cancer, gastric cancer, and breast cancer, and is linked to 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 such a variety of cancers, and various approaches are being attempted to inhibit c-MET expression and activity. However, as mentioned above, since c-METs are associated with the development and progression of various cancers, there is a persistent need for the development of novel therapeutic agents that target c-METs and can treat cancer.

[0020] Specifically, in the present invention, the anti-c-MET antibody is an antibody comprising a light chain variable region including the light chain CDR1 described in SEQ ID NO: 1; the light chain CDR2 described in SEQ ID NO: 2; and the light chain CDR3 described in SEQ ID NO: 3, and a heavy chain variable region including the heavy chain CDR1 described in SEQ ID NO: 4; the heavy chain CDR2 described in SEQ ID NO: 5; and the heavy chain CDR3 described in SEQ ID NO: 6.

[0021] In this invention, the term "heavy chain" can encompass the entire full-length heavy chain and its fragments, including a variable region domain VH containing an amino acid sequence having a sufficiently variable region sequence for conferring specificity to an antigen, and three constant region domains CH1, CH2, and CH3. Furthermore, the term "light chain" in this invention can encompass the entire full-length light chain and its fragments, including a variable region domain VL containing an amino acid sequence having a sufficiently variable region sequence for conferring specificity to an antigen, and a constant region domain CL.

[0022] In the present invention, the antibody may include all mouse antibodies produced from mice and mutants obtained by substituting, adding, and / or deleting parts of the amino acid sequence of the parent antibody in order to improve the affinity, immune function, etc. The mutants are not limited to these, but examples include chimeric antibodies, humanized antibodies, affinity-optimized antibodies, etc.

[0023] In the present invention, the term "mutant" broadly refers to an antibody in which a portion of the CDR amino acid sequence of the parent antibody is mutated (substituted, added, or deleted) under conditions that include the same CDR as the parent antibody or target the same epitope. Such mutants can be appropriately modified by those skilled in the art to improve the affinity and immunogenicity of the antibody while maintaining its ability to bind to the same epitope.

[0024] In other words, the antibody or antigen-binding fragment of the present invention may include not only the sequence of the anti-c-MET antibody described herein, but also its biological equivalents, to the extent that it can specifically recognize c-MET. For example, further modifications can 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, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side-chain substitutions, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side-chain substitutions reveals 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; as well as phenylalanine, tryptophan, and tyrosine, can be said to be biologically functional equivalents.

[0025] In this invention, the term "chimeric antibody" refers to an antibody in which the variable region of a mouse antibody and the constant region of a human antibody have been rearranged, resulting in an antibody that exhibits a significantly improved immune response compared to a mouse antibody.

[0026] In this invention, the term "humanized antibody" refers to an antibody whose protein sequence, derived from a non-human species, has been modified to resemble that of naturally occurring antibody variants produced by humans. As an example, a humanized antibody can be produced by combining a mouse-derived CDR with a human antibody-derived FR to create a humanized variable region, and then combining this with the constant region of a preferred human antibody. However, simply grafting a CDR results in low affinity for the humanized antibody. Therefore, by making several important FR amino acid residues that are thought to affect the three-dimensional structure of the CDR compatible with those of the mouse antibody, it is possible to achieve an affinity level similar to that of the original mouse antibody.

[0027] In this invention, the term "affinity-optimized antibody" refers to a mutant antibody in which a portion of the CDR sequence of a specific antibody is substituted, added, or deleted, resulting in an antibody that binds to the same antigen epitope as the specific antibody but with improved binding affinity to the antigen. Specifically, the affinity-optimized antibody of this invention refers to a mutant antibody that binds to the same epitope as an antibody containing a light chain variable region including the light chain CDR1 described in SEQ ID NO: 1; the light chain CDR2 described in SEQ ID NO: 2; and the light chain CDR3 described in SEQ ID NO: 3, and a heavy chain variable region including the heavy chain CDR1 described in SEQ ID NO: 4; the heavy chain CDR2 described in SEQ ID NO: 5; and the heavy chain CDR3 described in SEQ ID NO: 6. A person with the skills of an ordinary technician can produce the affinity-optimized antibody using known techniques based on specific light and heavy chain CDR sequences. For example, the affinity-optimized antibody of this invention can be produced via phage display. In this invention, the term "phage display" refers to a technique for displaying a mutant polypeptide as a fusion protein with at least a portion of the outer protein on the surface of a phage, such as a fibrous phage particle. The usefulness of phage display lies in its ability to rapidly and efficiently classify sequences that bind to target antigens with high affinity in large libraries of randomized protein variants. Displaying peptide and protein libraries on phages has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.

[0028] As one embodiment of the present invention, the antibody may include a light chain variable region described in SEQ ID NO: 7 and a heavy chain variable region described in SEQ ID NO: 8. For example, the antibody may include a light chain variable region encoded by a nucleotide described in SEQ ID NO: 9 and a heavy chain variable region encoded by a nucleotide described in SEQ ID NO: 10, but is not limited thereto.

[0029] As another embodiment of the present invention, the antibody may include (a) the light chain variable region described in SEQ ID NO: 11 and the heavy chain variable region described in SEQ ID NO: 12; or (b) the light chain variable region described in SEQ ID NO: 15 and the heavy chain variable region described in SEQ ID NO: 16. For example, the antibody may include, but is not limited to, (a) the light chain variable region encoded by the nucleotide described in SEQ ID NO: 13 and the heavy chain variable region encoded by the nucleotide described in SEQ ID NO: 14; or (b) the light chain variable region encoded by the nucleotide described in SEQ ID NO: 17 and the heavy chain variable region encoded by the nucleotide described in SEQ ID NO: 18. The antibody may also include the hinge region described in any of SEQ ID NOs: 19 to 26.

[0030] As yet another embodiment of the present invention, but not limited thereto, an affinity-optimized antibody for the antibody is an antibody comprising a light chain variable region including light chain CDR1 described in SEQ ID NO: 1; light chain CDR2 described in SEQ ID NO: 2; light chain CDR3 described in SEQ ID NO: 3; and a heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 4; heavy chain CDR2 described in SEQ ID NO: 5; heavy chain CDR3 described in SEQ ID NO: 6, wherein at least one amino acid sequence is substituted, and (i) light chain CDR (ii) The 1st position G is replaced with A, E, K, L, N, R, S, V or W; the 2nd position A is replaced with C, G, I, P, S, T or V; the 3rd position S is replaced with G, M, N, P, Q, R, S or T; the 4th position E is replaced with A, D, F, G, H, K, M, Q, R, S, T or V; the 5th position N is replaced with A, D, E, G, K, L, P, Q, R, S, T or V; the 6th position I is replaced with A, F, L, M, Q, R, S, T or V; the 7th position Y is replaced with F, H, R or V; or the 8th position G is replaced with D, F, H, M, N, R, S, T or V; (ii) light (iii) The G at position 1 of the chain CDR2 is replaced with D, F, H, K, P, Q, S, V, or Y; the T at position 3 is replaced with Q; or the N at position 4 is replaced with G; (iii) The Q at position 1 of the light chain CDR3 is replaced with E, G, I, M, or N; the N at position 2 is replaced with A, D, E, H, L, Q, S, or T; the V at position 3 is replaced with I, L, M, N, Q, S, or T; the L at position 4 is replaced with F, H, I, M, R, S, V, W, or Y; the S at position 5 is replaced with C, D, E, F, G, H, K, L, N, Q, R, T, V, or Y; and the S at position 6 is replaced with D, E, F, G, H, I, L, M, N, P , Q, R, T, V or Y; P at position 7 is replaced with A, D, E, G, N, Q, S or V; Y at position 8 is replaced with E, F, L, M or Q; or T at position 9 is replaced with D, F, G, I, L, N, S, V, W or Y; (iv) D at position 1 of heavy chain CDR1 is replaced with G or Q; Y at position 2 is replaced with Q; or I at position 4 is replaced with A or Q; (v) F at position 3 of heavy chain CDR2 is replaced with D, E, W or Y; G at position 5 is replaced with D, H or Y; S at position 6 is replaced with F, P, W or Y; G at position 7 is replaced with A, F, L, N or T; N at position 8 is F, P, S, T or Y; T in the 9th position is A, D, E, F, G, H, L, P, S or V; H in the 10th position is A, D, F, M, R, S, T, V, W or Y; F in the 11th position is G, H, I, L, M, N, P, Q, V or Y; S in the 12th position is A, D, G, H, I, L, P, T or V;The 13th position A is replaced with D, E, F, G, H, I, K, L, M, P, R, S, T, V or Y; the 14th position R is replaced with A, E, G, H, L, N, P, Q, S, W or Y; the 15th position F is replaced with D, E, G, L, M, P, R, S, V or W; the 16th position K is replaced with A, E, F, G, H, L, R, S, T, V or Y; or the 17th position G is replaced with E, F, H, L, M, N, P, Q, R, S, T, V or W; or (vi) the 1st position G of heavy chain CDR3 is replaced with E, F, H, N, Q, V, or W; D at position 2 is E; Y at position 3 is L, Q, T, or V; G at position 4 is W; F at position 5 is L or Y; L at position 6 is Q, S, or Y; or Y at position 7 is C, L, M, N, or Q, where light chain CDR1 may contain 0-5 substitutions, light chain CDR2 0-1 substitutions, light chain CDR3 0-7 substitutions, heavy chain CDR1 0-1 substitutions, heavy chain CDR2 0-11 substitutions, and heavy chain CDR3 0-6 substitutions.

[0031] As yet another embodiment of the present invention, the affinity-optimized antibody may specifically include a light chain variable region comprising: a light chain CDR1 described in SEQ ID NO: 1 and any of SEQ ID NOs: 124 to 163; a light chain CDR2 described in SEQ ID NOs: 2 and any of SEQ ID NOs: 164 to 174; and a light chain CDR3 described in SEQ ID NOs: 3 and any of SEQ ID NOs: 175 to 284; and a heavy chain CDR1 described in SEQ ID NOs: 4 and any of SEQ ID NOs: 36 to 40; a heavy chain CDR2 described in SEQ ID NOs: 5 and any of SEQ ID NOs: 41 to 110; and a heavy chain variable region described in SEQ ID NOs: 6 and any of SEQ ID NOs: 111 to 123; more specifically, a light chain variable region described in SEQ ID NOs: 11 and any of SEQ ID NOs: 289 to 294; and a heavy chain variable region described in SEQ ID NOs: 12 and any of SEQ ID NOs: 285 to 288; and even more specifically, (a) described in SEQ ID NO: 11 (b) The light chain variable region and the heavy chain variable region described in Sequence ID No. 285; (c) The light chain variable region described in Sequence ID No. 293 and the heavy chain variable region described in Sequence ID No. 12; (d) The light chain variable region described in Sequence ID No. 291 and the heavy chain variable region described in Sequence ID No. 288; (e) The light chain variable region described in Sequence ID No. 289 and the heavy chain variable region described in Sequence ID No. 285; (f) The light chain variable region described in Sequence ID No. 290 and the heavy chain variable region described in Sequence ID No. 286; (g) The light chain variable region described in Sequence ID No. 291 and the heavy chain variable region described in Sequence ID No. 287; (h) The light chain variable region described in Sequence ID No. 292 and the heavy chain variable region described in Sequence ID No. 287; (i) The light chain variable region described in Sequence ID No. 294 and the heavy chain variable region described in Sequence ID No. 287; or (j) The light chain variable region described in Sequence ID No. 289 and the heavy chain variable region described in Sequence ID No. 285, but are not limited thereto.

[0032] The antibody of the present invention is particularly characterized by being modified to include the terminal GlcNAc(N-acetylglucosamine) portion of the following formula 2. [ka]

[0033] By deforming the antibody to have the terminal GlcNAc portion at a specific position, it can easily bind to a desired number of drug-linker complexes, thus enabling the stable production of immune complexes.

[0034] The modified antibody contains an asparagine residue at positions 290-305 (e.g., N297; EU numbering), where a terminal GlcNAc portion may be present. This terminal GlcNAc portion may be linked to the antibody via the C1 position of the GlcNAc.

[0035] In the present invention, S is a sugar portion and includes all sugars or sugar derivatives. The sugars or sugar derivatives are not limited to, but may include galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc), or sialic acid (N-acetylneuraminic acid), and are all included in the scope of the present invention, at least within an equal range, as long as they can achieve the same objectives as the present invention. In the present invention, antibody modification can be achieved by 1) trimming the glycan structure of the antibody by endoglycosidase treatment (trimming), and 2) attaching the terminal GlcNAc portion to the antibody by treating it with nucleoside monophosphate or diphosphate-S-N3 together with glycosyltransferase (enzymatic remodeling). Through this method, an azide group can be introduced into the antibody (azido-Ab), and the modified antibody can be readily bound to a drug-linker complex by a nonmetallic click reaction, allowing for the easy production of an immune complex (Figure 2). The type of endoglycosidase and glycosyltransferase used for antibody modification in the present invention is not limited, and those skilled in the art can select and use an appropriate enzyme for antibody modification.

[0036] In the above equation 2, a may be 0 or 1, that is, fucose (Fuc) may not be present, or one may be added to GlcNAc.

[0037] The nucleoside monophosphate- or diphosphate used in antibody modification in the present invention may be, but is not limited to, UDP (uridine diphosphate), GDP (guanosine diphosphate), TDP (thymidine diphosphate), CDP (cytidine diphosphate), or CMP (cytidine monophosphate).

[0038] In the present invention, the sugar derivative S-N3 is, 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- It may also be 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 to these. Furthermore, if a reactive functional group other than an azide group, such as a keto or alkynyl group, is substituted, it should be understood that all such substitutions fall within the scope of the present invention as long as they exhibit effects equivalent to those of the present invention.

[0039] As described above, enzymatic remodeling can theoretically occur at only two positions in a single monoclonal antibody. Therefore, in principle, in this invention, it can be considered that there are two bindings between the antibody and BCN, thereby easily producing an immune complex with a 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 biantibody, the number of bindings may differ depending on the structure and form of the antigen-binding fragment or multivalent antibody. That is, in formula 1 above, Ab is bound to BCN-S U -L U -(C L The statement that two -D)p molecules are bound is based on the monoclonal antibody and may be interpreted differently depending on the structure and morphology of Ab.

[0040] In the present invention, the terminal azide group of the terminal GlcNAc moiety in Ab can bind to a BCN (Bicyclo[6.1.0]nonyne) ring or a derivative thereof to form a triazole moiety, thus enabling the production of the immune complex by a simple nonmetallic click reaction with a drug-linker complex. In the present invention, the BCN ring or a derivative thereof may be represented by the following formulas 7-1 to 7-3. [ka] In equations 7-1 to 7-3 above, X is -CR D -or -N-, R C These are independently -H, or -C as a substitution or non-substitution. 1-6 Alkyl, -C 3-10 Cycloalkyl, -3 to 10 member heterocycloalkyl, -C 6-12 They are aryl or -5 to 12-membered heteroaryls. R D is -H, -halo, or -C 1-3 It is alkyl, Ry is independently -halo, -OH, and -OC. 1-3 Alkyl, -NO2, -CN, -S(O)2C1-3 Alkyl, -C 1-6 Alkyl, -C 3-10 Cycloalkyl, -3 to 10 member heterocycloalkyl, -C 6-12 They are aryl or -5 to 12-membered heteroaryls. m is an integer between 1 and 4.

[0041] For example, the BCN ring may be bicyclo[6.1.0]nona-4-yne, in which case it forms the triazole moiety of formula 3 below. [ka] (Here, * is linked to the S of the antibody, and ** is linked to S U (Connected to) In the immune complex of the present invention, S U (Stretcher Unit) consists of a BCN ring and L U A linking group is used, and those skilled in the art can use linker units known in the art to appropriately set the distance between Ab and the cytotoxic drug moiety in the immune complex of the present invention. U Examples include -CH2-, -OC(=O)-, -C(=O)O-, and -NR E -, -O-, -S-, -CH2CH2O-, -OCH2CH2-, -NR F (C=O)-, -C(=O)NR G Functional groups such as -, -C(=O)-, -S(=O)-, and -S(=O)2- may be included (where R E , R F , R G These are independently -H or C 1-3 Any substance (that is alkyl) that can achieve the same objectives as the immune complex of the present invention is included within the scope of this invention.

[0042] As an example, in the immune complex of the present invention, S U This may be represented by the following formula 4. [ka]

[0043] Here, * is linked to the BCN ring, and ** is L U It is connected to, R A and R B These are independently -H or -C 1-3 It is alkyl, n is an integer between 1 and 5.

[0044] In the immune complex of the present invention, L U (Linking Unit) is the S U and C L This is the part that connects L, and may be a single link or a branched linker depending on the purpose. That is, a person skilled in the art will know U The Drug-Antibody Ratio (DAR) of the immune complex of the present invention can be adjusted according to its morphology, and accordingly, p is an integer from 1 to 3.

[0045] For example, but not limited to L U When linking two drugs via L, U L may be expressed by the following formula 5, U If three drugs are linked via L U This may be represented by the following formula 8. [ka] [ka] Here, * is S U It is linked to, and ** is C L It is connected to, R H is -H or -C 1-3 It is alkyl, M1 to M3 are each independently -C(=O)- or -OC(=O)-, q1 to q3 are each independent integers between 2 and 4.

[0046] In the immune complex of the present invention, C L A Cleavable Linker is a linker that can be cleaved, and may include linkers that can be cleaved enzymatically or non-enzymatically. For example, C L The peptide portion may include a peptide portion, which is a unit consisting of one or more natural or non-natural amino acids, and the immune complex of the present invention is cleaved in the body and contributes to the release of a drug. For example, if the peptide portion is a dipeptide, it may be, but is not limited to, -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-. L This may further include spacer units such as substituted or unsubstituted PABC (p-aminobezyloxy-carbonyl) groups. The PABC groups may also be substituted with sugars or derivatives thereof to form glucuronide units (see WO2007 / 011968).

[0047] As a specific example, C L The linker, which can be cut, may be represented by the following formula 6. [ka] (Here, Dipeptide is -Val-Cit-, -Val-Ala-, -Phe-Lys- or -Glu-Ala-, Each Rx is independently -C 1-3 Selected from alkyl, -halo, or -OH, r is an integer between 0 and 4.

[0048] In the immune complex of the present invention, D refers to a cytotoxic drug portion, which is a cytotoxic drug that can achieve a desired anticancer effect by being released intracellularly or around cancer cells after the immune complex of the present invention has entered cancer cells. The cytotoxic drug portion may be, but is not limited to, i) a DNA binding agent, 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, viiii) a NAMPT inhibitor, ix) a protein synthesis inhibitor, or x) an immunomodulatory compound.

[0049] As a specific example, in the immune complex of the present invention, the cytotoxic drug portion may be an auristatin compound, a calicheamicin compound, or an anthracycline compound. When these compounds are produced as an immune complex with the antibody or antigen-binding fragment of the present invention, they exhibit significantly superior anticancer activity compared to other c-MET-targeted ADCs known to date. In the present invention, the auristatin compound refers to auristatin and its derivative compounds that have cytotoxicity as tubulin inhibitors, and may, but are not limited to, auristatin, drastatin, MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), auristatin F, AF-HPA, MMAF-HPA, or AFP (phenylenediamine). In the present invention, the calicheamicin compound refers to calicheamicin derived from Micromonospora echinospora that binds to the DNA minor groove and exhibits cytotoxicity, and its derivative compounds. In the present invention, the anthracycline compound refers to a tetracyclic compound derived from Streptomyces peucetius that is inserted between DNA base pairs and exhibits cytotoxicity, and its derivative compounds.

[0050] In this invention, "stereoisomer" means a compound having the same chemical or molecular formula but being stereochemically different. Stereoiomers as used herein include optical isomers, enantiomers, diasteromers, cis / trans isomers, rotamers, and atropisomers, and each of these isomers, racemes, and mixtures thereof are also within the scope of this invention. Unless otherwise stated, solid line bonds linked to one chiral carbon atom ( [ka] ) is a wedge-shaped solid line connection that indicates the absolute arrangement of the center of the solid. [ka] ) or wedge-shaped dotted line connection ( [ka] ) can include

[0051] In this invention, "pharmaceutically acceptable salt" refers to salts commonly used in the pharmaceutical industry, including, for example, salts of inorganic ions such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, and iron, and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid. In addition, there are 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, orotic acid, and acetylsalicylic acid, as well as amino acid salts such as lysine, arginine, and guanidine. Furthermore, there are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium, which can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts referred to in this invention are not limited by these listed salts.

[0052] II. Pharmaceutical Compositions Another aspect of the present invention for achieving the above objective is a pharmaceutical composition for the prevention or treatment of cancer, comprising the immune complex, its stereoisomer, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0053] Another aspect of the present invention is a method for treating or preventing cancer, comprising the step of administering a therapeutically effective amount of the immune complex to an individual (subject) in need.

[0054] Another aspect of the present invention is the use of the immune complex for cancer prevention or treatment. Another aspect of the present invention is the use of the immune complex for the production of the pharmaceutical composition.

[0055] The aforementioned immune complexes, their stereoisomers, and pharmaceutically acceptable salts thereof are as described above.

[0056] The immune complex of the present invention specifically binds to c-MET and induces cancer cell death, and therefore can be usefully used for the treatment or prevention of cancer.

[0057] In the present invention, the cancer may be a solid tumor or a hematological cancer. For example, pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial carcinoma, ovarian germ cell carcinoma, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic leukemia Pharyngeal leukemia, retinoblastoma, choroidal melanoma, ampullary carcinoma of Vater, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, mast cell tumor, nasal and paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, childhood brain cancer, childhood 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 It may be one or more of the following: cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, ocular cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary origin, gastric lymphoma, gastric cancer, gastric carcinoid tumor, 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 tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, hematological cancer, and thymic cancer.

[0058] The aforementioned cancer may be a c-MET positive cancer and may include all cancers associated with the c-MET gene.

[0059] In this invention, the term "prevention" means any action that suppresses or delays the progression of cancer by administering the composition according to the present invention, and "treatment" means suppressing the onset of cancer, or reducing or eliminating cancer.

[0060] The term "therapeutably effective amount" as used in this invention refers to the amount of the immune complex effective in treating or preventing cancer. Specifically, "therapeutably effective amount" means an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including individual species and severity, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors widely known in the medical field. The pharmaceutical compositions of this invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and when administered in combination, they can be administered sequentially or simultaneously. They can also be administered single or multiple times. Taking all of the above factors into consideration, it is important to administer the amount that provides the maximum effect with the minimum amount without side effects, and the dosage can be easily determined by those skilled in the art depending on a variety of factors such as the patient's condition, age, sex, and comorbidities.

[0061] Embodiments of the present invention can be modified in several other forms, and the scope of the invention is not limited to the embodiments described below. Furthermore, embodiments of the present invention are provided to more fully explain the invention to a person of average skill in the art. Moreover, throughout this specification, the term "includes" a component does not exclude other components unless specifically stated otherwise, but rather means that other components may be further included. [Examples]

[0062] The configuration and effects of the present invention will be described in more detail below through examples. These embodiments are merely illustrative of the present invention, and the scope of the present invention is not limited by these embodiments.

[0063] Example 1. Production of hybridoma cells that produce c-MET-specific antibodies and confirmation of their tumor cell proliferation inhibitory activity. (1) Preparation and selection of hybridoma cell lines that produce monoclonal antibodies against c-MET protein. To obtain immunized mice necessary for developing hybridoma cell lines through animal immunization, human c-MET Sema domain / Fc fusion protein (developed in-house) was injected intraperitoneally into mice as an antigen. To select hybridoma cells that specifically react only to the c-MET protein from the hybridoma cell population, screening was performed using ELISA analysis with human c-MET / His fusion protein as the antigen.

[0064] (2)c-MET antibody Table 1 shows the light chain and heavy chain CDR amino acid sequences of the mouse antibody mCM05 obtained from selected hybridoma cell lines.

[0065] [Table 1]

[0066] (3) In vitro tumor cell proliferation inhibitory activity of hybridoma c-MET antibody The tumor cell proliferation inhibitory activity of c-MET-specific mouse antibodies (mCM05) obtained using hybridoma cell lines, and chimeric antibodies (cCM05-IgG1, cCM05-IgG2) prepared by fusing the aforementioned antibody with human heavy chain and light chain constant regions, was tested using the human glioblastoma cell line U-87MG and the human gastric cancer cell line MKN45.

[0067] Specifically, U-87MG cells (ATCC, #HTB14) were diluted in EMEM culture medium (ATCC, #30-2003) containing 10% (v / v) FBS, 100 U / 400 ml penicillin, and 100 μg / 500 ml streptomycin (Invitrogen, #15140-122), and 2.5 × 10⁶ cells were added to each well of a 96-well plate. 3After adding 100 μl of individual cell concentration to each well, the plates were cultured for 18–24 hours at 37°C, 95% relative humidity, and 5% (v / v) CO2. After removing the cell culture medium from each well, 100 μl of EMEM medium containing 2% (v / v) FBS was added to each well. Antibodies prepared at 2× final concentration (100 nM) were serially diluted by 1 / 10 and added to each well in 100 μl each at six concentrations (i.e., 200 nM, 20 nM, 2 nM, 200 pM, 20 pM, and 2 pM). Subsequently, the plates were cultured for 5 days at 37°C, 95% relative humidity, and 5% (v / v) CO2, and on the final day, the cells were fixed with 10% TCA (Trichloroacetic acid; Sigma, #T0699) solution. The fixed cells were stained with 80 μl of 0.4% SRB (sulforhodamine B) solution in each well for 25 minutes, and then washed five times with 1% acetic acid solution. After drying, 150 μl of 10 mM Tris solution was added to each well of the plate to dissolve the SRB dye, and the absorbance was measured at a wavelength of 540 nm using a microplate reader.

[0068] Additionally, the MKN45 (#JCRB0254) cell line was diluted in RPMI-1640 medium (Gibco, #A10491) containing 10% (v / v) FBS, and 2.5 × 10¹⁶ cells were placed in each well of a 96-well plate. 3After dispensing the cells individually and placing them in the plate, they were cultured overnight at 37°C under 5% CO2 conditions. Then, the culture medium in each well of the plate was replaced with 100 μl of RPMI-1640 medium containing 1% (v / v) FBS. The test antibody was then sequentially diluted by 1 / 10 increments 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 100 μl of each solution was added to each well. Next, the plate was cultured at 37°C under 5% CO2 conditions for 5 days, after which the medium was removed, and 200 μl of TCA solution was added to each well to fix the cells. The cells in the plate were stained according to a standard SRB colorimetric assay method, similar to the test for U87MG cells, and the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader. The results for the U87MG and MKN45 cell lines are shown in Table 2.

[0069] [Table 2]

[0070] As can be seen in Table 2 above, the anti-c-MET antibodies of the present invention have comparable or superior tumor cell proliferation inhibitory activity compared to the known c-MET antibodies LY2875358 and OA-5D5 (control group).

[0071] Table 3 below shows the specific consensus sequences for the light chain and heavy chain variable regions of the antibody of the present invention.

[0072] [Table 3]

[0073] Example 2. Production of a humanized antibody of mCM05 antibody and confirmation of its in vitro tumor cell proliferation inhibitory activity. To further confirm the effects of the antibody produced according to the present invention, as an example, the mouse antibody mCM05 was humanized, and its tumor cell proliferation inhibitory activity was confirmed in vitro.

[0074] To design the humanized mCM05 antibody heavy chain, we first analyzed human germline genes with high homology to the mCM05 antibody heavy chain variable region genes via Ig Blast (http: / / www.ncbi.nlm.nih.gov / igblast / ). As a result, we confirmed that IGHV3-23 has 48% homology to the mCM05 antibody at the amino acid level, and that IGHV3-11 has 46% homology to the mCM05 antibody at the amino acid level.

[0075] The CDR-H1, CDR-H2, and CDR-H3 of the mCM05 antibody were defined using Kabat numbering, and huCM05-1 was constructed by designing the CDR portion of the mCM05 antibody to be incorporated into the IGHV3-23 framework. At this time, amino acids 48 (V→I), 49 (S→G), 71 (R→A), 73 (N→K), 78 (L→A), and 94 (K→G) were back-mutated to the amino acid sequence of the original mCM05 antibody, and the heavy chain of huCM05-1 was finally constructed. In the case of huCM05-2, the CDR portion of the mCM05 antibody was designed to be incorporated into the IGHV3-11 backbone. Amino acids 48 (V→I), 49 (S→G), 71 (R→A), 73 (N→K), 78 (L→A), and 94 (R→G) were back-mutated to the amino acid sequence of the original mCM05 antibody, ultimately constructing the heavy chain of huCM05-2.

[0076] In the case of the mCM05 antibody light chain, for humanization design, human germline genes with high homology to the variable region genes of the mCM05 antibody light chain were analyzed using Ig Blast. As a result, it was confirmed that IGKV1-27 has 65.3% homology to the mCM05 antibody at the amino acid level, and IGKV1-33 has 64.2% homology to the mCM05 antibody at the amino acid level.

[0077] The CDR-L1, CDR-L2, and CDR-L3 of the mCM05 antibody were defined using Kabat numbering. HuCM05-1 was created by designing the CDR portion of the mCM05 antibody to be incorporated into the IGKV1-33 skeleton, and huCM05-2 was created by designing the CDR portion to be incorporated into the IGKV1-27 skeleton. In this process, both huCM05-1 and huCM05-2 underwent back-mutation of amino acid 69 (T→R) to the amino acid sequence of the original mCM05 antibody.

[0078] The CM05 humanized antibody was expressed in 293T cells using the pCLS05 vector (Registered Patent No. 10-1420274 in the Republic of Korea). The tumor cell proliferation inhibitory activity of the humanized antibody obtained in this way was confirmed in the human glioblastoma cell line U87MG using the same method as in Example 1.

[0079] As a result, the ICs huCM05-1 and huCM05-2 50 The values ​​were 30 nM and 24.6 nM, respectively, confirming that the antibody exhibits anticancer activity at a similar level to that of the chimeric cCM05-IgG1 antibody (IC50 = 32.4 nM).

[0080] Table 4 shows the specific consensus sequences for the light chain and heavy chain variable regions of the huCM05-1 and huCM05-2 humanized antibodies.

[0081] [Table 4A] [Table 4B]

[0082] Example 3. Preparation of hinge mutants and testing of their tumor cell proliferation inhibitory activity. Next, we performed tumor cell proliferation inhibitory activity tests according to the hinge sequence of the human IgG1 heavy chain constant region.

[0083] First, the hinge of the human IgG1 heavy chain constant region had the amino acid sequence "EPKSCDKTHTCPPCP (SEQ ID NO: 19)". By substituting this, a hinge region mutant having the amino acid sequences of SEQ ID NOs. 20 to 26 was obtained. This was then cloned into vectors containing the heavy chain variable regions of the huCM05-1 and huCM05-2 humanized antibodies prepared in Example 2. The tumor cell proliferation inhibitory activity by the hinge sequence was confirmed in vitro using U-87MG in the same manner as in Example 1.

[0084] Furthermore, the effects of huCM05-1 and huCM05-2 humanized antibodies on the non-small cell lung cancer cell line NCI-H1993 (ATCC, #CRL-5909) were analyzed as follows: The NCI-H1993 cell line was diluted in RPMI-1640 medium (Gibco, #A10491) containing 10% (v / v) FBS, and 3.0 × 10¹⁴ cells were placed in each well of a 96-well plate. 3 After dispensing the cells individually and placing them in the wells, they were incubated overnight at 37°C under 5% CO2 conditions. Subsequently, the culture medium in each well of the plate was replaced with 100 μl of RPMI-1640 medium containing 2% (v / v) FBS. The test antibody was then sequentially diluted by 1 / 10 increments (i.e., 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM) to a final concentration of 0.001 nM, and 100 μl of each solution was added to each well. The plate was then incubated at 37°C under 5% CO2 conditions for 5 days. After the medium was removed, 200 μl of TCA solution (Sigma, #T0699) was added to each well to fix the cells. The cells in the plate were stained according to a standard SRB colorimetric assay method, and the absorbance of each well was measured at a wavelength of 540 nm using a microplate reader.

[0085] The results for huCM05-1 in U-87MG and NCI-H1993 (ATCC, #CRL-5909) are shown in Table 5.

[0086] [Table 5]

[0087] As can be seen in Table 5, there are some differences in the tumor cell proliferation inhibitory activity of huCM05-1 antibodies due to differences in the hinge sequence, but it was confirmed that most effectively inhibit the proliferation of tumor cells. Therefore, in the following, we named the IgG1 humanized antibody with the hinge region of SEQ ID NO: 20 in relation to huCM05-1 as huCM05, created an affinity-optimized antibody against it, and confirmed its effect.

[0088] Example 4. Preparation of affinity-optimized antibodies for huCM05 and confirmation of their in vitro tumor cell proliferation inhibitory activity. To produce an affinity-optimized antibody for huCM05, a phage-displayed scFv library was first prepared using a phagemide vector in which scFv and pIII were conjugated. The approximate structure of the vector is shown in Figure 1. The phagemide vector contained scFv sections of the antibody under the control of an IPTG-inducible lac promoter, and the linker sequence used was GGGGS GGGGS GGGGS (SEQ ID NO: 35).

[0089] Next, to introduce diversity into the heavy and light chain CDR sites of huCM05, mutagenic oligonucleotides containing NNK codons were used. This resulted in the creation of huCM05 scFv libraries fused with His, HA, and pIII, and antibodies specific to human c-MET were selected from the resulting antibody library.

[0090] Specifically, a competitive selection method was used to select antibodies with improved affinity. Human c-MET antigen was conjugated to Dynabeads® M-280 (Thermo Fisher Scientific, 11205D) according to the manufacturer's guidelines. The antigen-conjugated beads were blocked with Superblock Tris buffered saline (Pierce) for 2 hours. Recombinant phages were grown overnight at 37°C, then centrifuged, and the supernatant phages were blocked with Superblock TBS and 0.05% Tween20 for 2 hours. The beads were then washed with PBS containing 0.05% Tween20. The blocked phage solution was added to the washed beads, incubated in a rotator for 2 hours for phage binding, and then washed with PBS containing 0.05% Tween20. Subsequently, human c-MET antigen was added to 1 ml of PBS containing 0.05% Twin 20 and incubated in a rotator for 24 hours (Rouet R et al. (2012) Nat Protoc. 7:364-373). Next, the phages bound to the beads were eluted with 100 mM triethanolamine for 5 minutes, and the eluent was neutralized with 0.5 M Tris / Cl (pH 7.2). The neutralized eluted phage solution was used to infect E. coli TG1.

[0091] Individual clones selected from the above experiments were grown in 200 μl of 2xYT broth supplemented with carbenicillin and ampicillin in a 96-well format. The culture supernatant was directly used in ELISA to select phage-displayed scFvs that bound to plates coated with the target protein. 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 amino acid sequences of the variable regions of representative light and heavy chain affinity-optimized antibodies are shown in Table 8.

[0092] [Table 6A] [Table 6B] Table 6C Table 6D Table 6E

Table 6F

Table 6G

[0093] Table 7A Table 7B Table 7C Table 7D Table 7E Table 7F

Table 7G

[0094] Table 8

[0095] In vitro growth inhibitory activity tests were performed on the U-87MG cell line using a portion of the affinity-optimized antibody, and the results are shown in Table 9.

[0096] [Table 9]

[0097] As confirmed in Table 9, the tumor cell proliferation inhibitory activity of the huCM05 affinity-optimized antibody in U-87MG cells was observed in IC 50 The efficacy was 5.0 to 18 nM, and compared to the parent antibody huCM05, the efficacy was increased by 4.3 to 9.8 times. The above results were performed on a portion of the antibodies having the amino acid sequences presented in Tables 6 to 8, but the affinity of the parent huCM05 antibody was optimized. These were all selected based on antigen affinity during the selection process, and it is expected that the remaining affinity-optimized antibodies, as well as antibodies combining the presented heavy chain and light chain variable region CDRs, will have a sufficiently similar effect.

[0098] For further experiments, ten affinity-optimized antibodies were created by combining the light chain and heavy chain variable regions described above. Specific combinations of light chain and heavy chain sequences are shown in Table 10.

[0099] [Table 10]

[0100] Next, the tumor cell proliferation inhibitory activity was evaluated using the same method as in Example 1, and the results are shown in Table 11.

[0101] [Table 11]

[0102] As confirmed in Table 11 above, not only huCM05 but also the 10 major antibodies, which combine the light chain and heavy chain variable regions of its affinity-optimized antibodies, similarly showed tumor cell proliferation inhibitory activity. In particular, the above 10 antibodies showed IC 50 The concentration was 1.7 to 5.3 nM, confirming that it has a tumor cell proliferation inhibitory effect 9.2 to 28.5 times better than the parent antibody huCM05.

[0103] Example 5. Measurement of binding force to ECD (BIAcore) Next, to measure the binding affinity of the c-MET antibody of the present invention to the extracellular domain (ECD), the binding of the c-MET antibody to human and cynomolgus monkey c-MET ECDs was measured using BIAcore, using human c-MET ECDs (ACROBiosystems, MET-H5227) and cynomolgus monkey c-MET ECDs (Sino Biological, 90304-C08H).

[0104] First, to capture anti-c-MET antibodies, Fc-specific anti-Human IgG antibodies (SouthernBiotech, 2047-01) were immobilized on CM5 sensor tips at a level of 10,000 RU. The antibodies were diluted to a concentration of 1-2 μg / ml 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) and injected at a flow rate of 30 μl / min for 10-120 seconds into the CM5 tips immobilized with anti-Human Ig Fc, followed by capture in the range of 150-200 RU. Each antigen was used after dilution to 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM, and injected sequentially from the lowest concentration. Subsequently, the vias were injected at a flow rate of 30 μl / min for 5 minutes to induce binding, and then dissociated by injecting running buffer for 10–15 minutes. The tips were 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 of BIAevaluation software version 4.1, and the via core data are summarized in Table 12.

[0105] [Table 12]

[0106] The aforementioned data demonstrates that the huCM05 antibody of the present invention binds to human and cynomolgus monkey c-MET ECD with excellent affinity.

[0107] Example 6. Cross-reactivity of c-MET antibodies against various cell surface receptors The specificity of the huCM05 antibody that specifically binds to c-MET according to the present invention, and its cross-reactivity to other receptor tyrosine kinase antigens, were analyzed using an indirect ELISA method. Among the major receptor tyrosine kinases, five antigens were selected for analysis: FGF R3, VEGFR R2, IGF IR, PDGF R, and RON.

[0108] In this example, the following antigens were used: 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).

[0109] 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), then coated overnight at 4°C. The plate was washed once with TBS-T, and to inhibit nonspecific binding, 200 μl of 4%-skim milk-containing TBS-T was added to each well and reacted at 37°C for 1 hour. After washing once with TBS-T buffer, the primary antibody was sequentially diluted in 2%-skim milk-containing TBS-T buffer from a maximum concentration of 30 nM to 0.002 nM, and 100 μl was added to each well and reacted at 37°C for 2 hours. After washing three times with TBS-T buffer, anti-human kappa light chains-peroxidase (Sigma, A7164) was diluted 1:5000 with a secondary antibody and 100 μl was added to each well. The mixture was then incubated at 37°C for 1 hour. After washing three times with TBS-T buffer, 100 μl of TMB solution (Sigma, T4444) was added to each well to allow the color reaction to proceed. Subsequently, 50 μl of 2N ammonium sulfate solution was added to each well to interrupt the reaction. Absorbance was measured using a microplate reader at a wavelength of 450 nm, with a reference wavelength of 570 nm. The degree of binding of anti-c-MET antibody to each antigen was proportional to the absorbance signal value, and the results are shown in Table 13.

[0110] [Table 13]

[0111] As confirmed in Table 13, the huCM05 antibody of the present invention specifically binds to c-MET and has been confirmed to hardly bind to other antigens such as FGF R3, VEGFR R2, IGF IR, PDGF R, and RON.

[0112] Example 7. Production of c-MET-ADC (Antibody-Drug Conjugate) (1) Overview of manufacturing Synaffix's GlycoConnectTM Using the technology, an ADC containing the anti-c-MET antibody of the present invention was produced. GlycoConnect TM The technology involves attaching azide groups to the glycosylation sites of antibodies via enzymatic remodeling and producing ADCs (Animal Derived Cells) through a simple click reaction (Figure 2). Since each heavy chain of the antibody has one glycosylation site, this method has the advantage of being able to uniformly produce ADCs with the desired drug-antibody ratio (DAR).

[0113] An ADC was produced by reacting huCM05, the anti-c-MET antibody of the present invention, with a linker-payload complex (Formula 9) containing MMAE, and named CM05-Auri. [ka]

[0114] The ADC of the present invention is produced through a two-step process: enzymatic remodeling of the antibody and drug attachment. The manufacturing process of CM05-Auri (CM05-BCN-HS-(vc-PABC-MMAE)2) is shown in Figure 3. Briefly, to remodel the antibody, endoglycosidase, glycosyltransferase (GalNAc-transferase), UDP-sugar, and alkaline phosphatase are added to the antibody and reacted. The remodeled antibody is then mixed with a linker-payload complex and bound by a metal-free click chemistry reaction to produce the ADC.

[0115] (2) Glycan remodeling The huCM05 antibody was added to TBS (pH 7.5) to a concentration of 25 mg / ml, and endoglycosidase, glycosyltransferase, UDP-sugar, and MnCl2 were added. The mixture was then incubated overnight at 30°C. The mixture was purified with Protein A to remove glycan remodeling components and added to PBS (pH 7.4) to a concentration of 25 mg / ml. After overnight incubation, the samples were degraded with IdeS and then subjected to MS analysis (Figure 4). The results confirmed that the huCM05 antibody was remodeled in a form modified with an azide group (azido-huCM05).

[0116] (3) Linker-payload coupling A azido-huCM05 antibody (15 mg / ml, PBS pH 7.4) and a linker-payload complex containing MMAE (BCN-HS-(vc-PABC-MMAE)2) were reacted in 7 equivalents of the linker-payload complex (i.e., 3.5 equivalents per azido) in a co-solvent under 10% DMF conditions and incubated overnight at room temperature.

[0117] Next, the samples cut with IdeS were examined by MS analysis (Figure 5), and it was confirmed that the desired ADC was successfully generated for each payload (Figure 6).

[0118] As a comparative example, Teliso-V (Telisotuzumab vedotin) was prepared with reference to International Publication No. 2017 / 201204, and B12-MMAE (BCN-HS-(va-PABC-MMAE)2 conjugation of B12 antibody, which is an anti-gp120 antibody) was prepared by the same method as described above.

[0119] (5) Analysis of ADC characteristics Table 14 shows the results of analyzing the characteristics of the ADCs in the examples and comparative examples of the present invention.

[0120] [Table 14]

[0121] Example 8. Testing of antigen binding affinity of c-MET-ADC (1) Evaluation of target binding using SPR (Surface Plasmon Resonance) The binding affinity of CM05-Auri and the naked antibody (huCM05) was compared using an SPR-equipped system (BIAcore T200). Anti-human Fc antibody (SouthernBiotech) was immobilized on a CM5 sensor chip, and the antibody or ADC was captured on it. Binding and dissociation times were then measured using the target antigen protein.

[0122] As a result, the K of huCM05 and CM05-Auri against c-MET D The values ​​were shown as 462 pM and 442 pM, respectively (Table 15). This indicates that the binding ability to the target protein does not change even when huCM05 is converted to CM05-Auri.

[0123] [Table 15]

[0124] (2) Evaluation of target protein binding affinity using ELISA (Enzyme-Linked Immunosorbent Assay) The binding activity of CM05-Auri and Teliso-V to the target protein c-MET was compared using ELISA analysis. CM05-Auri or Teliso-V was added to plates coated with recombinant c-MET protein, and the binding level was evaluated.

[0125] As a result, CM05-Auri targeted EC 50 The molecular weight was 0.2 nM, which demonstrated superior target binding ability compared to Teliso-V, which showed 0.65 nM (Figure 7).

[0126] (3) Evaluation of binding affinity to target protein-expressing cells using FACS (Fluorescence-activated Cell Sorting) Binding affinity was analyzed using NCI-H441 and NCI-H1975 cell lines expressing c-MET at high and intermediate levels, respectively. After incubating cells with CM05-Auri or Teliso-V, a fluorescent secondary antibody was attached, and the ADCs bound to the cells were measured using FACS analysis.

[0127] As a result, in the NCI-H441 cell line, Teliso-V-bound EC 50 While the combined EC of CM05-Auri is 0.67 nM, 50 The binding affinity was shown at 0.15 nM, indicating better binding affinity than Teliso-V. Experiments using the NCI-H1975 cell line also showed that CM05-Auri binding EC 50 The binding affinity was 0.32 nM, which was superior to that of Teliso-V (1.43 nM) (Figure 8).

[0128] Example 9.c Internalization analysis of MET-ADC (1) Evaluation of the internalization rate To confirm whether the internalization rate is maintained when the huCM05 antibody is converted to ADC, the internalization rates of the naked antibody (huCM05) and CM05-Auri were compared. After incubation of HT-29 cells expressing the target protein and ADC together, unbound ADC was removed, and internalization was induced at 37°C. Subsequently, the amount of ADC that entered the cells was measured by FACS at each time point (every 30 minutes, up to a maximum of 2 hours).

[0129] As a result, the internalization rate of ADC was similar to that of huCM05 antibody, and it was confirmed that there was no change in the internalization rate even when huCM05 antibody was converted to CM05-Auri (Figure 9).

[0130] (2) Evaluation of continuous internalization To measure the amount of ADC entering cells through internalization, a continuous internalization assay was performed. H441 and H1975 cell lines were treated with ADC, incubated at 37°C, and then the amount of ADC entering cells over time was measured by FACS.

[0131] As a result, it was confirmed that CM05-Auri entered cells in greater quantities than Teliso-V in all of the cell lines tested. Six hours after treatment with H441 and H1975, CM05-Auri showed values ​​1.6 times and 1.9 times higher than Teliso-V, respectively (Table 16 and Figure 10).

[0132] [Table 16]

[0133] Since ADCs primarily exhibit cytotoxic activity after internalization, the experimental results described above suggest that CM05-Auri has superior efficacy compared to Teliso-V.

[0134] Example 10. Evaluation of the in vitro efficacy of c-MET-ADC (1) Evaluation of in vitro efficacy using cancer cell lines First, 100 μl of cells were seeded into a 96-well plate at the indicated density. The plate was incubated in a humidified incubator at 37°C under 5% CO2 conditions for 24 hours. Next, serial dilutions of CM05-Auri, Teliso-V, or MMAE-free drugs were prepared using culture medium containing 10% FBS (test agents). After removing the culture medium from the 96-well plate, 200 μl of the prepared test agent was added to each plate containing cells. The plates were then incubated in a humidified incubator at 37°C under 5% CO2 conditions for 6 days. After removing the culture medium, 100 μl of 10% TCA fixative solution was added to each well, and the plate was incubated at 4°C 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 the plates were stained at room temperature for 30 minutes. The plates were washed three times by immersing them in a tub filled with 0.1% acetic acid, and then air dried at room temperature. Subsequently, 150 μl of 10 mM Trizma base solution was added to each well, and the plates were left at room temperature for 30 minutes to allow the protein-binding dye to dissolve. The absorbance was then measured using a microplate reader at 540 nm.

[0135] Cell proliferation was calculated as follows:

[0136] Using statistical analysis with Prism software and the curve-fitting method, a dose-response curve between concentration and cell proliferation percentage was plotted, and the EC 50 The maximum cell death value (percentage of cell proliferation inhibition with 1 μg / ml ADC) was calculated. The approximate number of c-MET molecules on the cell surface was determined by FACS analysis using QIFIKIT (Agilent) as antibody-binding capacity to the c-MET antibody.

[0137] As a result, CM05-Auri's EC 50In cell lines with c-MET expression levels of 29,000 or higher, all of these were shown at sub-nanomole levels, and the EC was lower than that of Teliso-V. 50 It was found that CM05-Auri exhibited more potent efficacy than Teliso-V in vitro (Table 17). On the other hand, in cell lines that did not express c-MET, neither of the two ADCs showed efficacy, which suggests that the efficacy of ADCs is dependent on the target protein.

[0138] [Table 17]

[0139] (2) Evaluation of the bystander effect The bystander effect of CM05-Auri was confirmed using H441, a c-MET overexpressing cell line, and H520 and H661, cell lines that do not express c-MET. When H520 and H661 cells were treated with the same medium that had been treated with CM05-Auri for 5 days, cell proliferation was suppressed even in c-MET-negative cell lines (Figure 11). These results indicate that CM05-Auri releases a drug to cells expressing the target protein, and the released drug kills cells that do not express the target protein, suggesting that CM05-Auri has a bystander effect.

[0140] Example 11. Evaluation of the in vivo efficacy of c-MET-ADC (1) Study of the in vivo activity of CM05-Auri in the H441CDX model To evaluate the antitumor effects of CM05-Auri and huCM05 in an H441NSCLC xenograft model, BALB / c-nude mice were inoculated with H441 cells to induce tumor formation. The tumor size was approximately 200 mm. 3 When the mice reached a certain stage, they were grouped together and injected with the drug into their intraperitoneal cavity once a week for a total of four times.

[0141] On day 35, the tumor size in the CM05-Auri 1 mg / kg group was 62.45 mm. 3 The vehicle control group (760.08 mm) 3 ) and the huCM05 3 mg / kg dose group (692.62 mm 3 Compared to the other approach, tumor growth was significantly suppressed (Figure 12).

[0142] (2) Study of the in vivo activity of CM05-Auri in the H1975CDX model To evaluate the antitumor effects of CM05-Auri and huCM05 in an H1975NSCLC xenograft model, BALB / c-nude mice were inoculated with H1975 cells to induce tumor formation. The tumor size was approximately 200 mm. 3 When the mice reached a certain stage, they were grouped together and injected with the drug into their intraperitoneal cavity once a week for a total of four times.

[0143] On day 35, the tumor size in the CM05-Auri 3mg / kg group was 167.71 mm. 3 The vehicle control group (2318.13 mm) 3 ) and CM05 3 mg / kg dose group (2496.14 mm 3 Compared to the other approach, tumor growth was significantly suppressed (Figure 13).

[0144] (3) Study on the in vivo efficacy of CM05-Auri in the HT-29CDX model To evaluate the antitumor effects of CM05-Auri and huCM05 in an HT-29CRC xenograft model, HT-29 cells were inoculated into BALB / c-nude mice to induce tumor formation. The tumor size was approximately 110 mm. 3 When the mice reached a certain stage, they were grouped together and injected with the drug into their intraperitoneal cavity once a week for a total of four times.

[0145] On day 34, the tumor size in the CM05-Auri 3 mg / kg group was 175.72 mm. 3 The vehicle control group (2315.64 mm) 3) and the CM05 3 mg / kg administration group (2411.92 mm 3 ) showed that tumor growth was significantly inhibited when compared (Figure 14).

[0146] (4) Comparative study on the in vivo activities of CM05-Auri and Teliso-V in the HCC827CDX model To compare and evaluate the antitumor effects of CM05-Auri with 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 mm 3 , the mice were grouped and each drug was injected once into the intraperitoneal space.

[0147] On the 24th day, the tumor size in the CM05-Auri 4.5 mg / kg administration group was 261.74 mm 3 , and it was shown to be significantly decreased when compared with the vehicle control group (P<0.05). On the contrary, the tumor size in the Teliso-V 4.5 mg / kg administration group was 836.84 mm 3 , and no significant difference was observed when compared with the vehicle control group (Figure 15).

[0148] (5) Study on the comparison of in vivo activities of CM05-Auri and Teliso-V in the H1573CDX model To compare and evaluate the antitumor effects of CM05-Auri with 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 mm 3 , the mice were grouped and each drug was injected once into the intraperitoneal space.

[0149] On the 35th day, the tumor size in the CM05-Auri 9 mg / kg administration group was 475.39 mm 3 , and the vehicle control group (1859.50 mm 3 , P<0.05) and the Teliso-V 9 mg / kg administration group (954.74 mm 3It was shown to be significantly decreased compared to P < 0.05 (Figure 16).

[0150] (6) Study on the comparison of in vivo activities of CM05-Auri and Teliso-V in the LU11681 PDX model To compare and evaluate the antitumor effect of CM05-Auri with Teliso-V in the LU11681 NSCLC xenograft model, tumor pieces (2 - 3 mm in diameter) were subcutaneously inoculated into the right flank of NOD / SCID mice to form tumors. When the tumor size reached approximately 300 mm 3 each mouse was grouped and each drug was injected once into the intraperitoneal space.

[0151] On the 25th day, the tumor sizes in the CM05-Auri 4.5 mg / kg and 9 mg / kg administration groups were 1050.75 mm 3 and 590.44 mm 3 respectively, and were shown to be significantly decreased compared to the Teliso-V 4.5 mg / kg and 9 mg / kg administration groups (1932.13 mm 3 and 1428.05 mm 3 , P < 0.05) (Figure 17).

[0152] (7) Study on the comparison of in vivo activities of CM05-Auri and Teliso-V in the LU5165 PDX model To compare and evaluate the antitumor effect of CM05-Auri with Teliso-V in the LU5165 NSCLC xenograft model, tumor pieces (2 - 3 mm in diameter) were subcutaneously inoculated into the right flank of NOD / SCID mice to form tumors. When the tumor size reached approximately 300 mm 3 each mouse was grouped and each drug was injected once into the intraperitoneal space.

[0153] ​​​​​​and 1002.88mm 3 This was shown to be a significant decrease compared to (Figure 18).

[0154] Example 12. Blood stability test of c-MET-ADC To evaluate the stability of CM05-Auri, its blood stability was tested using cynomolgus monkeys. The results confirmed that CM05-Auri is very stable in cynomolgus monkeys (Figure 19). These results support the idea that CM05-Auri is also stable in humans.

[0155] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, the above-described embodiments should be understood to be illustrative and not limiting in all respects. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts described below, rather than from the above-described detailed description.

Claims

1. The immune complex represented by the following formula 1, its stereoisomer, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 In the above formula 1, Ab is an antibody comprising a light chain variable region including light chain CDR1 described in SEQ ID NO: 1; light chain CDR2 described in SEQ ID NO: 2; light chain CDR3 described in SEQ ID NO: 3; and a heavy chain variable region comprising heavy chain CDR1 described in SEQ ID NO: 4; heavy chain CDR2 described in SEQ ID NO: 5; heavy chain CDR3 described in SEQ ID NO: 6; an affinity-optimized antibody thereof; or an antigen-binding fragment thereof; The Ab includes the terminal GlcNAc portion of the following formula 2, 【Chemistry 2】 (Here, S is GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) as the sugar portion, Fuc is fucose. a is either 0 or 1); The terminal azide group of the terminal GlcNAc moiety in Ab is bonded to the BCN (Bicyclo[6.1.0]non-4-yne) ring, forming the triazole moiety of formula 3 below. 【Transformation 3】 (Here, * is linked to the S of the antibody, and ** is linked to S Ｕ (to be linked to); S Ｕ The BCN ring and L Ｕ The base connecting them is represented by the following equation 4, 【Chemistry 4】 (Here, * is connected to the BCN ring, and ** is L Ｕ It is connected to, R Ａ and R Ｂ These are independently -H or -C １－３ It is alkyl, n is an integer between 1 and 5); L Ｕ It is either a single bond or a branched linker. L Ｕ When L is a branched linker, Ｕ it is represented by the following formula 5, 【Transformation 5】 (Here, * is S Ｕ It is connected to, and ** is C Ｌ It is connected to, M １ and M ２ These are independently -C(=O)- or -OC(=O)-, q １ and q ２ (Each of these is an independent integer between 2 and 4); C Ｌ As a linker that can be cut, it is represented by the following equation 6: 【Transformation 6】 (Here, Dipeptide is -Val-Cit-, -Val-Ala-, -Phe-Lys- or -Glu-Ala-, Each Rx is independently -C １－３ Selected from alkyl, -halo, or -OH, (where r is an integer between 0 and 4); D is the cytotoxic drug portion; and p is an integer between 1 and 3.

2. The immunocomplex according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Ab comprises the light chain variable region described in SEQ ID NO: 7 and the heavy chain variable region described in SEQ ID NO:

8.

3. The immunocomplex according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Ab comprises a hinge region as described in any of SEQ ID NOs. 19 to 26.

4. The affinity-optimized antibody is An antibody comprising a light chain variable region including light chain CDR1 described in SEQ ID NO: 1; light chain CDR2 described in SEQ ID NO: 2; light chain CDR3 described in SEQ ID NO: 3; and a heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 4; heavy chain CDR2 described in SEQ ID NO: 5; and heavy chain CDR3 described in SEQ ID NO: 6, wherein at least one amino acid sequence is substituted. (i) The G at position 1 of the light chain CDR1 is replaced with A, E, K, L, N, R, S, V or W; the A at position 2 is replaced with C, G, I, P, S, T or V; the S at position 3 is replaced with G, M, N, P, Q, R, S or T; the E at position 4 is replaced with A, D, F, G, H, K, M, Q, R, S, T or V; the N at position 5 is replaced with A, D, E, G, K, L, P, Q, R, S, T or V; the I at position 6 is replaced with A, F, L, M, Q, R, S, T or V; the Y at position 7 is replaced with F, H, R or V; or the G at position 8 is replaced with D, F, H, M, N, R, S, T or V; (ii) The G at position 1 of the light chain CDR2 is replaced by D, F, H, K, P, Q, S, V or Y; the T at position 3 is replaced by Q; or the N at position 4 is replaced by G; (iii) The Q at position 1 of the light chain CDR3 is replaced with E, G, I, M or N; the N at position 2 is replaced with A, D, E, H, L, Q, S or T; the V at position 3 is replaced with I, L, M, N, Q, S or T; the L at position 4 is replaced with F, H, I, M, R, S, V, W or Y; the S at position 5 is replaced with C, D, E, F, G, H, K, L, N, Q, R, T, V or Y; the S at position 6 is replaced with D, E, F, G, H, I, L, M, N, P, Q, R, T, V or Y; the P at position 7 is replaced with A, D, E, G, N, Q, S or V; the Y at position 8 is replaced with E, F, L, M or Q; or the T at position 9 is replaced with D, F, G, I, L, N, S, V, W or Y; (iv) The D at position 1 of the heavy chain CDR1 is replaced with G or Q; the Y at position 2 is replaced with Q; or the I at position 4 is replaced with A or Q; (v) The F at position 3 of the heavy chain CDR2 is D, E, W or Y; the G at position 5 is D, H or Y; the S at position 6 is F, P, W or Y; the G at position 7 is A, F, L, N or T; the N at position 8 is F, P, S, T or Y; the T at position 9 is A, D, E, F, G, H, L, P, S or V; the H at position 10 is A, D, F, M, R, S, T, V, W or Y; the F at position 11 is G, H, I, L, M, N, P, Q, V or Y; the S at position 12 is A, D, G, H, I, L, P, T, or V; A at position 13 is replaced with D, E, F, G, H, I, K, L, M, P, R, S, T, V, or Y; R at position 14 is replaced with A, E, G, H, L, N, P, Q, S, W, or Y; F at position 15 is replaced with D, E, G, L, M, P, R, S, V, or W; K at position 16 is replaced with A, E, F, G, H, L, R, S, T, V, or Y; or G at position 17 is replaced with E, F, H, L, M, N, P, Q, R, S, T, V, or W; or (vi) The G at position 1 of the heavy chain CDR3 is replaced with E, F, H, N, Q, V, or W; the D at position 2 is replaced with E; the Y at position 3 is replaced with L, Q, T, or V; the G at position 4 is replaced with W; the F at position 5 is replaced with L or Y; the L at position 6 is replaced with Q, S, or Y; or the Y at position 7 is replaced with C, L, M, N, or Q. The immunocomplex according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein light chain CDR1 contains 0 to 5 substitutions, light chain CDR2 contains 0 to 1 substitution, light chain CDR3 contains 0 to 7 substitutions, heavy chain CDR1 contains 0 to 1 substitution, heavy chain CDR2 contains 0 to 11 substitutions, and heavy chain CDR3 contains 0 to 6 substitutions.

5. The affinity-optimized antibody is A light chain variable region including: light chain CDR1 described in SEQ ID NO: 1 and any of SEQ ID NOs: 124 to 163; light chain CDR2 described in SEQ ID NO: 2 and any of SEQ ID NOs: 164 to 174; and light chain CDR3 described in SEQ ID NO: 3 and any of SEQ ID NOs: 175 to 284, and The immunocomplex according to claim 1, comprising: a heavy chain CDR1 as described in SEQ ID NO: 4 and any of SEQ ID NOs: 36 to 40; a heavy chain CDR2 as described in SEQ ID NO: 5 and any of SEQ ID NOs: 41 to 110; and a heavy chain variable region as described in SEQ ID NO: 6 and any of SEQ ID NOs: 111 to 123; a stereoisomer thereof; or a pharmaceutically acceptable salt thereof.

6. The affinity-optimized antibody is The immunocomplex according to claim 1, comprising a light chain variable region described in SEQ ID NO: 11 and any of SEQ ID NOs: 289 to 294, and a heavy chain variable region described in SEQ ID NO: 12 and any of SEQ ID NOs: 285 to 288, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

7. The affinity-optimized antibody is (a) The light chain variable region described in Sequence ID No. 11 and the heavy chain variable region described in Sequence ID No. 285; (b) The light chain variable region described in Sequence ID No. 11 and the heavy chain variable region described in Sequence ID No. 288; (c) The light chain variable region described in Sequence ID No. 293 and the heavy chain variable region described in Sequence ID No. 12; (d) The light chain variable region described in Sequence ID No. 291 and the heavy chain variable region described in Sequence ID No. 288; (e) The light chain variable region described in Sequence ID No. 289 and the heavy chain variable region described in Sequence ID No. 285; (f) The light chain variable region described in Sequence ID No. 290 and the heavy chain variable region described in Sequence ID No. 286; (g) The light chain variable region described in Sequence ID No. 291 and the heavy chain variable region described in Sequence ID No. 287; (h) The light chain variable region described in Sequence ID No. 292 and the heavy chain variable region described in Sequence ID No. 287; (i) the light chain variable region described in Sequence ID No. 294 and the heavy chain variable region described in Sequence ID No. 287; or (j) The immunocomplex according to claim 1, comprising the light chain variable region described in SEQ ID NO: 289 and the heavy chain variable region described in SEQ ID NO: 285, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

8. The immunocomplex according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug portion is an auristatin compound MMAE (Monomethyl auristatin E).

9. A pharmaceutical composition for the prevention or treatment of cancer, comprising an immune complex according to any one of claims 1 to 8, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

10. The pharmaceutically active composition for the prevention or treatment of cancer according to claim 9, wherein the cancer is a c-MET-positive cancer.

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