Anti-cMet Antibody, Antibody-Drug Conjugate, and Method for Preparing and Using the Same

Abstract

The present disclosure provides an antibody or an antigen-binding fragment thereof targeting c-MET, an antibody-drug conjugate, and their use in the treatment of cancer. The present disclosure further provides a nucleotide encoding the c-MET antibody or an antigen-binding fragment thereof, a combination of polynucleotides, an expression vector and a combination of expression vectors, a drug composition comprising the c-MET antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, and their applications in the preparation of a drug for treating or preventing cancer.

Description

Technical Field

[0001] The present application belongs to the field of pharmaceutical technology, and relates to various antibodies, antibody-drug conjugates and their preparation methods, and their use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to the prevention and / or treatment of tumor diseases.

Background Art

[0002] cMET is a tyrosine kinase receptor expressed on the cell membrane, binds to the ligand HGF via the Sema domain, thereby triggering a downstream phosphorylation cascade reaction, and ultimately exerting a promoting effect on cell proliferation. Currently, the target inhibitors of c-Met kinase mainly include three types: HGF and c-Met biological antagonists, HGF and c-Met antibodies, and small molecule c-Met inhibitors. Existing clinical results have shown that the therapeutic effects of antibodies directly corresponding to HGF and c-Met, or small molecule c-Met inhibition, are not ideal.

[0003] Antibodies that compete with HGF for binding usually cause dimerization of the MET receptor and thus act as agonists, making the development of therapeutic antibodies against MET difficult (Prat M, et al. J Cell Sci 1998; 111 (Pt 2), 237-247). Onartuzumab is the first anti-c-Met antibody developed and is derived from the humanized development of the c-Met agonist antibody 5D5.

[0004] Cytotoxic chemotherapy was once the standard treatment for cancer, but highly lethal cytotoxic molecules kill normal cells and cause severe toxic side effects. Targeted antitumor drugs have both targeting properties and antitumor activity, and thus have become the focus of current cancer research. However, due to the problem of target selectivity of targeted drugs, relatively large toxic side effects often occur, which limits the therapeutic effect of targeted drugs. Biopolymer drugs such as antibodies or antibody fragments have strong targeting properties, but their therapeutic effect on solid tumors is limited or non-existent. An ADC is a conjugate of an antibody and a small molecule drug, which fuses the targeting effect of the antibody and the activity of the bioactive molecule, and becomes a biological missile with highly anticipated therapeutic effects and safety advantages. The antibody guides the ADC to bind to target cells, and then it is endocytosed. The small molecule drug is released through enzymatic cleavage generated under the action of specific enzymes inside the cell to treat the disease.

[0005] Therefore, screening for monoclonal antibodies with high affinity, specifically recognizing cMet, and effectively blocking HGF binding and downstream signaling pathways is very important for the subsequent development of ADC drugs. Regarding the C-Met target, by developing differential, high-quality, and safe antibodies or ADC drugs, a wider range of excellent pharmaceutical options can be provided for cancer patients, and a broad market outlook can also be obtained.

Summary of the Invention

[0006] The present disclosure provides an antibody targeting c-MET or an antigen-binding fragment thereof, an antibody-drug conjugate, and their use in the treatment of cancer. The present disclosure further provides a nucleotide encoding the c-MET antibody or an antigen-binding fragment thereof, a combination of polynucleotides, an expression vector and a combination of expression vectors, a drug composition comprising the c-MET antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, and their applications in the preparation of drugs for treating or preventing cancer.

[0007] In a first aspect, the present disclosure provides an antibody targeting c-MET or an antigen-binding fragment thereof. Specifically, the present disclosure provides an anti-c-MET antibody or an antigen-binding fragment thereof. In some embodiments, the c-MET is human c-MET.

[0008] In some embodiments, the antibody or its antigen-binding fragment comprises three complementarity-determining regions (HCDR) from the heavy-chain variable region: HCDR1, HCDR2, and HCDR3, and / or three complementarity-determining regions (LCDR) from the light-chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy-chain variable region and / or a light-chain variable region. In some embodiments, the heavy-chain variable region comprises three complementarity-determining regions (CDR) from the heavy-chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light-chain variable region comprises three complementarity-determining regions (CDR) from the light-chain variable region: LCDR1, LCDR2, and LCDR3.

[0009] In some embodiments, the CDR of the heavy-chain variable region and / or the CDR of the light-chain variable region are identical to the CDR sequences of the antibodies specified by the following sequences, or have 1, 2, or 3 amino acid substitutions compared to the CDR of the antibodies specified by the following sequences. The antibodies specified by the sequences are as follows: (1) The amino acid sequence of the heavy-chain variable region is shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12, and / or, (2) The amino acid sequence of the light-chain variable region is shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11.

[0010] In some embodiments, the three complementarity-determining regions (HCDR) from the heavy-chain variable region of the present disclosure: HCDR1, HCDR2, and HCDR3 are (i) The three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12, or, (ii) For any one of the arrays in (i), the three HCDR regions are selected from arrays containing a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

[0011] Preferably, the HCDR is determined according to the AbM, Chothia, Kabat, Contact, or IMGT definition scheme.

[0012] In some embodiments, the three complementarity-determining regions (LCDR) from the light chain variable region of the present disclosure: LCDR1, LCDR2, and LCDR3 are (i) The three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, Or, (ii) For any one of the arrays in (i), the three LCDR regions are selected from arrays containing a total of at least one and no more than 5, 4, 3, 2, or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably, the LCDR is determined according to the AbM, Chothia, Kabat, Contact, or IMGT definition scheme.

[0013] In some embodiments, the anti-C-MET antibody or antigen-binding fragment thereof according to any one of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 10, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 9.

[0014] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 12, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 11.

[0015] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:2, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:1.

[0016] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:4, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:3.

[0017] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:6, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:5.

[0018] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:8, and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7.

[0019] In certain embodiments, HCDR1-3 and LCDR1-3 are identified using different measurement methods or systems or definition schemes.

[0020] In certain embodiments, HCDR1-3 and LCDR1-3 are identified according to the AbM, Chothia, Kabat, Contact, or IMGT definition schemes. In certain embodiments, the complementarity determining regions HCDR1-3 and LCDR1-3 are determined based on the corresponding heavy and light chain variable regions using the Chothia, Kabat, or IMGT definition schemes as shown in the sequences and their specific information tables.

[0021] In some embodiments, the HCDR1 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO:, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO: [Table 1]

[0022] In some embodiments, the HCDR2 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO:, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO: [Table 2]

[0023] In some embodiments, the HCDR2 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 38 or 61, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 38 or 61.

[0024] In some embodiments, the HCDR3 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO:, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

Table 3

[0025] In some embodiments, the LCDR1 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO:, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

Table 4

[0026] In some embodiments, the LCDR2 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO:, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

Table 5

[0027] In some embodiments, the LCDR3 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO:, or comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

Table 6

[0028] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein a. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 that respectively contain the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, or consists of the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 that respectively contain the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, or consists of the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the IMGT definition scheme.

[0029] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein b. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 that respectively contain the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 38 (QIRLKSLNYATHYAXSVKG, where X may be any amino acid such as E or Q), and SEQ ID NO: 19, or consists of the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 38 (QIRLKSLNYATHYAXSVKG, where X may be any amino acid such as E or Q), and SEQ ID NO: 19. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 that respectively contain the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, or consists of the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0030] In some embodiments, the anti-C-MET antibody according to any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein c. the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 each including the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19, or consisting of the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 each including the amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, or consisting of the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively, Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0031] In some embodiments, the anti-C-MET antibody according to any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein d. the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 each including the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19, or consisting of the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 each including the amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, or consisting of the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively, Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0032] In some embodiments, the anti-C-MET antibody according to any one of the above items includes a heavy chain variable region and / or a light chain variable region, where e. the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 each including the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 53, and SEQ ID NO: 19, or consisting of the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 53, and SEQ ID NO: 19 respectively, and the light chain variable region includes the amino acids shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22 respectively, or consists of the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22 respectively, Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Chothia definition scheme.

[0033] In some embodiments, the anti-C-MET antibody according to any one of the above items includes a heavy chain variable region and / or a light chain variable region, where f. the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 each including the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, or consisting of the amino acid sequences shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 each including the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 respectively, or consists of the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 respectively, Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the IMGT definition scheme.

[0034] In some embodiments, the anti-C-MET antibody according to any one of the above items includes a heavy chain variable region and / or a light chain variable region, where g. Each of the heavy chain variable regions comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:61 (WIFPGSGNTKYX1X2KFX3G, where X1, X2 and X3 may be any amino acid, for example, X1 is I or S, X2 is E or Q, and / or X3 is K or Q) and SEQ ID NO:32, or consists of the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:61 (WIFPGSGNTKYX1X2KFX3G, where X1, X2 and X3 may be any amino acid, for example, X1 is I or S, X2 is E or Q, and / or X3 is K or Q) and SEQ ID NO:32. Each of the light chain variable regions comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:35, or consists of the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:35. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0035] In some embodiments, the anti-C-MET antibody according to any one of the above comprises a heavy chain variable region and / or a light chain variable region, where h. Each of the heavy chain variable regions comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32, or consists of the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32. Each of the light chain variable regions comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:35, or consists of the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:35. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0036] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein, i. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, or consisting of the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 35, or consisting of the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 35 respectively. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0037] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein, j. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 32, or consisting of the amino acid sequences shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 32 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 35, or consisting of the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 35 respectively. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Chothia definition scheme.

[0038] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein, k. the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, or consisting of the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, or consisting of the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47 respectively, Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the IMGT definition scheme.

[0039] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein, l. the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, or consisting of the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each comprising the amino acid sequence shown in SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, or consisting of the amino acid sequences shown in SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50 respectively, Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme.

[0040] In some embodiments, the anti-C-MET antibody according to any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein m. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 each including the amino acid sequence shown in SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 44, or consisting of the amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 44, respectively. The light chain variable region includes LCDR1, LCDR2, and LCDR3 each including the amino acid sequence shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, or consisting of the amino acid sequences shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively. Preferably, the above HCDR1-3 and LCDR1-3 are determined according to the Chothia definition scheme.

[0041] In some embodiments, the three heavy chain CDRs contained in the anti-C-MET antibody or its antigen-binding fragment according to the present disclosure contain a total of 5 or fewer amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions) compared to the above respective sets of HCDR1-3. In some embodiments, the three light chain CDRs contained in the anti-C-MET antibody or its antigen-binding fragment according to the present disclosure contain a total of 5 or fewer amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions) compared to the above respective sets of LCDR1-3.

[0042] The anti-C-MET antibody or its antigen-binding fragment according to any one of the above items includes a heavy chain variable region and a light chain variable region, wherein 4, 3, 2, or 1 of the 6 CDRs of HCDR1-3 and LCDR1-3 contained in the heavy chain variable region and the light chain variable region perform 1, 2, or 3 amino acid substitutions. In some preferred embodiments, the substitutions are conservative substitutions.

[0043] In some embodiments, the anti-C-MET antibody according to any one of the above is a humanized antibody, a mouse antibody, or a chimeric antibody. In some embodiments, the antibody of the present disclosure is humanized. Humanization can be achieved by substituting one or more amino acid residues, particularly the framework region sequences, in the heavy chain variable region and the light chain variable region of a non-human native antibody with the residues at the corresponding positions in the variable regions of conventional human-derived antibodies. Methods for humanizing antibodies are well known in the art. Usually, humanization substitutions are performed in a manner that maintains the advantageous binding properties of the antibody. Tests for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art to determine and select appropriate humanizing residue mutations or combinations of mutations.

[0044] In some embodiments, the anti-C-MET antibody according to any one of the above is a monoclonal antibody. In some embodiments, the antigen-binding fragment of the c-MET antibody is selected from Fab, Fab’, Fab’-SH, F(ab’)2, Fv, or single-chain Fv (scFv).

[0045] In some embodiments, the heavy chain variable region of the present disclosure (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:2, 4, 6, 8, 10, or 12, or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:2, 4, 6, 8, 10, or 12, or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions of amino acids) compared to an amino acid sequence selected from SEQ ID NO:2, 4, 6, 8, 10, or 12, and preferably, the amino acid changes do not occur in the CDR regions.

[0046] In some embodiments, the light chain variable region of the present disclosure is (i) an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9 or 11 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of said amino acid sequence, or (ii) an amino acid sequence containing, or consisting of, an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9 or 11, or (iii) an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative substitutions of amino acids) compared to an amino acid sequence selected from SEQ ID NO: 2, 4, 5, 7, 10 or 11, or consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.

[0047] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein said heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10, and / or said light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9.

[0048] In some embodiments, the anti-C-MET antibody according to any one of the above items comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region contains, or consists of, the amino acid sequence shown in SEQ ID NO: 10, and said light chain variable region contains, or consists of, the amino acid sequence shown in SEQ ID NO: 9.

[0049] In some embodiments, the anti-C-MET antibody described in any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:12, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:11.

[0050] In some embodiments, the anti-C-MET antibody described in any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO:12, and the light chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO:11.

[0051] In some embodiments, the anti-C-MET antibody described in any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:2, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1.

[0052] In some embodiments, the anti-C-MET antibody described in any one of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO:1.

[0053] In some embodiments, the anti-C-MET antibody described in any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:4, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:3.

[0054] In some embodiments, the anti-C-MET antibody described in any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region includes, or consists of, the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region includes, or consists of, the amino acid sequence shown in SEQ ID NO:3.

[0055] In some embodiments, the anti-C-MET antibody described in any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:6, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:5.

[0056] In some embodiments, the anti-C-MET antibody described in any one of the above items includes a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region includes, or consists of, the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region includes, or consists of, the amino acid sequence shown in SEQ ID NO:5.

[0057] In some embodiments, the anti-C-MET antibody according to any one of the above has a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:8, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:7.

[0058] In some embodiments, the anti-C-MET antibody according to any one of the above has a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region comprises, or consists of, the amino acid sequence shown in SEQ ID NO:7.

[0059] In some embodiments, the anti-C-MET antibody according to any one of the above further comprises an antibody heavy chain constant region. In some embodiments, the anti-C-MET antibody according to any one of the above further comprises an antibody light chain constant region. In some embodiments, the anti-C-MET antibody according to any one of the above further comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 or IgG4 constant regions. In some embodiments, the light chain constant region is selected from human antibody κ or λ chain constant regions.

[0060] In some preferred embodiments, the antibody heavy chain constant region of the present disclosure is (i) comprises, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:51, (ii) comprises, or consists of, the amino acid sequence of SEQ ID NO:51, or (iii) an amino acid sequence having one or more (preferably 20 or less, more preferably 10 or less, still more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 51, or consisting of said amino acid sequence.

[0061] In some embodiments, said amino acid changes occur in the Fc region.

[0062] In some embodiments, the antibody light chain constant region of the present disclosure (i) includes, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 52, (ii) includes, or consists of, the amino acid sequence of SEQ ID NO: 52, or (iii) includes, or consists of, an amino acid sequence having one or more (preferably 20 or less, more preferably 10 or less, still more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 52.

[0063] In some embodiments, said antibody includes a heavy chain constant region and a light chain constant region, said heavy chain constant region includes, or consists of, the amino acid sequence shown in SEQ ID NO: 51, and said light chain constant region includes, or consists of, the amino acid sequence shown in SEQ ID NO: 52.

[0064] In some embodiments, the anti-C-MET antibody according to any one of the above further includes an antibody heavy chain. In some embodiments, the anti-C-MET antibody according to any one of the above further includes an antibody light chain. In some embodiments, the anti-C-MET antibody according to any one of the above further includes an antibody heavy chain and a light chain.

[0065] In some preferred embodiments, the antibody heavy chain of the present disclosure (i) an amino acid sequence selected from SEQ ID NO: 56 or 58, and comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 56 or 58, or (iii) comprising or consisting of an amino acid sequence having one or more (preferably 20 or fewer, more preferably 5, 4, 3, 2, 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 56 or 58.

[0066] In some embodiments, the antibody light chain of the present disclosure is (i) an amino acid sequence selected from SEQ ID NO: 57 or 59, and comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 57 or 59, or (iii) comprising or consisting of an amino acid sequence having one or more (preferably 20 or fewer, more preferably 5, 4, 3, 2, 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 57 or 59.

[0067] In some embodiments, the anti-C-MET antibody according to any one of the above items is It comprises a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:56, and / or a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:57.

[0068] In some embodiments, the anti-C-MET antibody according to any one of the above comprises a heavy chain and a light chain, the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:56, and the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:57.

[0069] In some embodiments, the anti-C-MET antibody according to any one of the above It comprises a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:58, and / or a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:59.

[0070] In some embodiments, the anti-C-MET antibody according to any one of the above comprises a heavy chain and a light chain, the heavy chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:58, and the light chain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:59.

[0071] In some embodiments, the present disclosure further provides an anti-C-MET antibody or an antigen-binding fragment thereof, wherein the antibody competitively binds to human C-MET with the anti-C-MET antibody described in any one of the above. As defined herein, an antibody that competitively binds to a reference antibody and its antigen means an antibody that blocks the binding of 50%, 60%, 70%, 80%, 90% or 95% or more of the reference antibody and the antibody in a competitive assay. Conversely, the reference antibody blocks the binding of 50%, 60%, 70%, 80%, 90% or 95% or more of the antibody and its antigen in a competitive assay. Many types of competitive binding assays can be used to determine whether an antibody competes with another antibody, and these assays include, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay, bioluminescence interference assay (e.g., Fortebio) or surface plasmon resonance (Biacore), etc.

[0072] In some embodiments, the anti-C-MET antibody or an antigen-binding fragment thereof competitively binds to human C-MET with the 45A5G10-Hz antibody.

[0073] In some embodiments, the anti-C-MET antibody or an antigen-binding fragment thereof competitively binds to human C-MET with the 55A10G6-Hz antibody.

[0074] In some embodiments, the anti-C-MET antibody or an antigen-binding fragment thereof competitively binds to human C-MET with both the 55A10G6-Hz antibody and the 45A5G10-Hz antibody.

[0075] In one embodiment of the present disclosure, the amino acid changes described herein include amino acid substitutions, insertions or deletions. In some embodiments, the amino acid changes are conservative changes. For a polypeptide sequence, "conservative change" includes substitutions, deletions or additions to the polypeptide sequence, but does not substantially change the desired functional activity of the polypeptide sequence.

[0076] Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. A conservative substitution means that one amino acid is replaced by another amino acid within the same class, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. For example, conservative substitutions always result in an amino acid being replaced by a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Eight sets of amino acids that include mutually conservative substitutions are shown below. 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); 8) Cysteine (C), Methionine (M). In some embodiments, the term "conservative sequence variation" is used to refer to amino acid modifications that do not significantly affect or alter the desired antigen-binding properties of the antibody molecules or binding protein molecules of the disclosure that contain the amino acid sequence. For example, a conservatively modified variant maintains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100 - 110% or more, of the binding affinity for the target antigen compared to the parental antibody or binding protein.

[0077] In preferred embodiments, the amino acid changes described in the present disclosure occur in regions outside the CDRs (e.g., within the FRs). More preferably, the amino acid changes described in the present disclosure occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0078] In certain embodiments, the substitution occurs in the CDR regions of the antibody. Typically, the resulting variant will have a modification (e.g., improvement) in certain biological properties (e.g., increased affinity) compared to the parental antibody and / or will have certain biological properties that are substantially retained from the parental antibody.

[0079] In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, such as "thioMAbs", where one or more residues of the antibody are replaced with cysteine residues.

[0080] In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Moieties suitable for antibody-directed action include, but are not limited to, water-soluble polymers.

[0081] In certain embodiments, the antibodies provided by the present disclosure are multispecific antibodies such as bispecific antibodies, trispecific antibodies or tetravalent antibodies.

[0082] In a second aspect, the present disclosure provides a multispecific binding molecule such as a multispecific antibody comprising the anti-C-MET antibody or antigen-binding fragment thereof described above. In certain preferred embodiments, said multispecific antibody is a bispecific antibody, a trispecific antibody or a tetravalent antibody.

[0083] In a third aspect, in some embodiments, the present disclosure further provides a nucleic acid molecule encoding the anti-C-MET antibody or fragment thereof described in any one of the above, or a nucleic acid of any one of its heavy or light chains.

[0084] For example, the nucleic acids of the present disclosure include a nucleic acid encoding an amino acid sequence set forth in any one selected from SEQ ID NOs: 1-12 and 56-59, or an amino acid sequence set forth in any one selected from SEQ ID NOs: 1-12 and 56-59 and having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. As will be apparent to those skilled in the art, due to codon degeneracy, the amino acid sequence of each antibody or polypeptide can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the molecules of the present disclosure can be generated using methods well known in the art such as novel solid-phase DNA synthesis or PCR amplification.

[0085] The heavy and / or light chains of the antibody molecules of the present disclosure can be fused at the N-terminus with a secretory signal peptide and / or a tag peptide that facilitates purification for production and purification.

[0086] The present disclosure further relates to vectors containing the nucleic acid, such as expression vectors such as eukaryotic expression vectors. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pTT5 vector such as the pTT5-mFc vector and the pTT5-hFc vector.

[0087] The present disclosure further provides a host cell containing the nucleic acid molecule or vector according to any one of the above. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., HEK293E or HEK293 cells)) or other cells applicable to the preparation of antibodies or fragments thereof. In one embodiment, the host cell is a prokaryote, such as a bacterium such as Escherichia coli.

[0088] The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Expression vectors can be constructed using methods well known to those skilled in the art. When an expression vector containing one or more nucleic acid molecules of the present invention for expression is prepared, the expression vector can be transfected or introduced into an appropriate host cell. To achieve this purpose, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, transduction with retroviruses, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0089] In a fourth aspect, the present disclosure provides a method for preparing an anti-c-MET antibody or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or the fragment thereof (preferably the antigen-binding fragment) or one or both strands thereof, and optionally isolating the antibody or the fragment thereof (preferably the antigen-binding fragment). In certain embodiments, the method further comprises recovering an anti-c-MET antibody or a fragment thereof (preferably an antigen-binding fragment) from the host cell.

[0090] Antibodies prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography such as protein A, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., which will be apparent to those skilled in the art.

[0091] In a fifth aspect, in some embodiments, the present disclosure further provides an immune complex (e.g., an antibody-drug conjugate) comprising the anti-C-MET antibody or an antigen-binding fragment thereof according to any one of the above and an effector molecule, wherein the effector molecule is bound to the anti-C-MET antibody or an antigen-binding fragment thereof, and preferably, the effector molecule is selected from a radioisotope, an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0092] In a sixth aspect, in some embodiments, the present disclosure further provides a method for using the antibody or an antigen-binding fragment thereof of the present invention for diagnosis and detection, and a composition for diagnosis and detection comprising the same.

[0093] In certain embodiments, any anti-c-MET antibody or an antigen-binding fragment thereof provided herein can be used to detect the presence of c-MET in a biological sample.

[0094] As used herein, the term "detect" includes quantitative or qualitative detection, and exemplary detection methods may relate to immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule-conjugated magnetic beads, ELISA assay, PCR technology (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum or other fluid sample derived from an organism. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is derived from tumor tissue or cancer tissue.

[0095] In one embodiment, an anti-c-MET antibody for a diagnostic or detection method is provided.

[0096] In another embodiment, a method for detecting the presence of c-MET in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of c-MET protein in the biological sample. In certain embodiments, c-MET is human c-MET or cynomolgus c-MET. In certain embodiments, the method comprises contacting the biological sample with the anti-c-MET antibody described herein under conditions that allow binding of the anti-c-MET antibody to c-MET, and detecting whether a complex is formed between the anti-c-MET antibody and c-MET. The formation of a complex indicates the presence of c-MET. This method may be an in vitro method or an in vivo method. In one embodiment, the anti-c-MET antibody is used to select a subject suitable for treatment with the anti-c-MET antibody. For example, c-MET is a biomarker used to select the subject. In some embodiments, the method is performed in vitro or in vivo.

[0097] In certain embodiments, a labeled antibody or fragment thereof is provided. Labels include labels or moieties that are directly detectable (e.g., fluorescent labels, chromophore labels, high electron density labels, chemiluminescent labels and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, by enzyme reactions or molecular interactions, but are not limited thereto.

[0098] In some embodiments provided by this specification, the sample is obtained before treatment with the antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained before using other treatment methods. In some embodiments, the sample is obtained during or after treatment with other treatment methods.

[0099] In some embodiments, c-MET is detected before treatment, for example, before starting treatment, or before a specific treatment after a treatment interval.

[0100] In some embodiments, a method for treating a disease of the present invention is provided, the method comprising examining the presence of c-MET in a subject (e.g., a sample) (e.g., a subject sample), thereby determining a c-MET value, comparing the c-MET value with a control value (e.g., the value in a normal individual), and when the c-MET value is greater than the control value, administering to the subject an antibody or fragment thereof or an antibody-drug conjugate, pharmaceutical composition, formulation, combination product, etc. described in the present invention, optionally in combination with one or more other treatment methods in a therapeutically effective amount, thereby treating the disease.

[0101] Accordingly, in one embodiment, the present disclosure further provides a method for immuno-detection or measurement of C-MET, the method comprising contacting an anti-C-MET antibody described in any one of the above with a subject or a sample from the subject.

[0102] In a seventh aspect, the present disclosure further provides an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof or a pharmaceutically acceptable solvate thereof, comprising the antibody or antigen-binding fragment thereof described in any one of the above.

[0103] In some embodiments, the structure of the antibody-drug conjugate is represented by formula (I),

Chemical formula

[0104] In some embodiments, the q is selected from 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 2 to 8 or 4 to 6, for example, selected from integers of 1 to 10.

[0105] Preferably, the q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0106] In some embodiments, the L covalently binds to an amino residue or a thiol residue of the antibody Ab. Preferably, the L covalently binds to a thiol residue of the antibody Ab. More preferably, the L covalently binds to a sulfhydryl residue formed after the inter-chain disulfide bond of the antibody Ab is opened.

[0107] In some embodiments, the L is a cleavable linker or a non-cleavable linker. Preferably, L is a cleavable linker.

[0108] In some embodiments, the cleavable linker contains a peptide unit, the peptide unit contains 2 to 10 amino acid residues, and the amino acid residues are selected from natural amino acid residues, unnatural amino acid residues, or the amino acid residues shown in AA 1 or its stereoisomers,

[0109] In some embodiments, the peptide unit contains at least one (for example, 1, 2 or 3) AA 1A dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide containing the amino acid residue represented by [[ID=]] or its stereoisomer.

[0110] In some preferred embodiments, in some embodiments, the peptide unit is one AA 1 A dipeptide, tripeptide or tetrapeptide containing the amino acid residue represented by [[ID=]] or its stereoisomer.

[0111] In some embodiments, the peptide unit consists of the following amino acids, i. At least one (e.g., one, two or three) AA 1 The amino acid residue represented by [[ID=]] or its stereoisomer, and ii. At least one natural amino acid residue and / or at least one unnatural amino acid residue.

[0112] AA 1 The structure of the amino acid residue shown in [[ID=]] is as follows,

Chemical formula

Chemical formula

[0113] Preferably, the amino acid residue is selected from -Val-, -Ala-, -Gly-, -Cit-, -AA 1 -, -Arg-, -Phe-, -Lys- and -Asn-.

[0114] Preferably, the peptide unit is -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -valine-lysine- (-Val-Lys-), -valine-arginine- (-Val-Arg-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylalanine-lysine- (-Phe-Lys-), -phenylalanine-arginine- (-Phe-Arg-), -alanine-alanine-alanine- (-Ala-Ala-Ala-), -alanine-alanine-asparagine- (-Ala-Ala-Asn-), -valine-AA 1 -glycine- (-Val-AA 1 -Gly-), -valine-AA 1 -alanine- (-Val-AA 1 -Ala-), -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-) and -glycine-glycine-valine-alanine- (-Gly-Gly-Val-Ala-).

[0115] In some preferred embodiments, the L is

Chemical formula

[0116] In some embodiments, L 1 is [Chemical formula] selected from, position 1 is linked to Tb via an S atom, and position 2 is linked to L 2 or L 3 linked to.

[0117] In some embodiments, L 1 is [Chemical formula] selected from Position 1 is linked to Tb via an S atom, and position 2 is linked to L 2 or L 3 is linked.

[0118] In some embodiments, L 1 is [Chemical formula] selected from Position 1 is linked to Tb via an S atom, and position 2 is linked to L 2 or L 3 is linked.

[0119] In some embodiments, L 1 is [Chemical formula] selected from Position 1 is linked to Tb via an S atom, and position 2 is linked to L 2 or L 3 is linked.

[0120] L 2 is absent or present, and when L 2 is present, L 2 is [Chemical formula] selected from each y1 is selected from any integer from 1 to 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer from 0 to 10 (e.g., 6 to 10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, position 1 is linked to L 1 is linked, and position 2 is linked to L 3 is linked, In some embodiments, L2 either does not exist or exists, and L 2 if exists, then L 2 is

Chem.

[0121] In some embodiments, L 2 either does not exist or exists, and L 2 if exists, then L 2 is

Chem.

[0122] In some embodiments, L 2 either does not exist or exists, and L 2 if exists, then L 2 is

Chem.

[0123] In some embodiments, L 2 does not exist.

[0124] In some embodiments, L 2 is

Chem.

[0125] L 3 is

Chem.

Chem.

[0126] In some preferred embodiments, the

Chem.

Table 7

[0127] Preferably, the

Chem.

Table 8-1

Table 8-2

Table 8-3

Table 8-4

[0128] In some embodiments, the active drug unit is selected from cytotoxic agents. In some embodiments, the active drug unit is a DNA topoisomerase inhibitor (e.g., camptothecin, DXD, camptothecin with modified substituents or DXD with modified substituents, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, such as camptothecin-based bioactive molecules), or a microtubulin inhibitor (e.g., MMAF-type microtubulin inhibitor, MMAE-type microtubulin inhibitor).

[0129] In some embodiments, the antibody-drug conjugate is a compound of formula (IIA-1) or formula (IIA-2),

Chemical formula

Chemical formula

Chemical formula

[0130] In some embodiments, the antibody-drug conjugate is a compound of formula (IIB-1) or formula (IIB-2),

Chemical formula

Chemical formula

Chemical formula

[0131] In some embodiments, the antibody-drug conjugate has the following structure

Table 9-1

Table 9-2

Table 9-3

[0132] Here, Ab and q are as defined above.

[0133] In an eighth aspect, the present disclosure provides a method for preparing an antibody-drug conjugate targeting C-MET, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof, wherein the antibody-drug conjugate has a structure represented by Formula I, and the method includes the following steps: (1) React an anti-c-MET antibody or a fragment thereof with a reducing reagent in a buffer solution to obtain a reduced antibody or a fragment thereof. Preferably, the reducing reagent is a disulfide reducing agent, such as TCEP. Preferably, the buffer solution has a pH of 6.0 to 8.0, such as a pH of 6.5, 7.0, 7.5, or 8.0, and more preferably is a phosphate buffer solution. (2) Crosslink a drug linker (linker-drug conjugate) with the reduced antibody or a fragment thereof obtained in step (1) in a mixed solution of a buffer solution and an organic solvent to obtain an antibody-drug conjugate targeting c-MET. Here, the buffer solution is as defined above. Preferably, the organic solvent is selected from dimethyl sulfoxide.

[0134] In some embodiments of the present disclosure, the c-MET antibody or a fragment thereof is as defined above, and the drug linker has a structure represented by the following Formula (IIIA-1), (IIIA-2), (IIIB-1), or (IIIB-2):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0135] In some embodiments, the drug linker has a structure of the following formula

Table 10-1

Table 10-2

[0136] The drug linker (Linker-Payload) of the present disclosure can be prepared by various methods known in the art, for example, it can be obtained by chemical synthesis techniques. The linker payload in the above antibody-drug conjugate can be prepared with reference to WO22022170971 and then can be conjugated to form an ADC with an antibody.

[0137] In a ninth aspect of the present disclosure, the present disclosure provides a group of antibody-drug conjugates, which include, or consist of, the antibody-drug conjugate described in the seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers or its pharmaceutically acceptable solvates, wherein the antibody-drug conjugate has one, two or more q values.

[0138] In some embodiments, when one q value of the antibody-drug conjugate in the group of antibody-drug conjugates occupies a majority (80%, 85%, 90%, 95%, 95%, 97%, 98%, 99%), the q value is close to the average DAR.

[0139] In some embodiments, when there is only one antibody-drug conjugate with a q value in the group of antibody-drug conjugates, the q value is equal to the average DAR.

[0140] In some embodiments, when the antibody-drug conjugates in the group of antibody-drug conjugates have two or more q values, the ratio of all the antibody-drug conjugates in the composition of the antibody-drug conjugate having one specific q value among them is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%.

[0141] In some embodiments, the average ratio of drug to antibody (average DAR) in the antibody-drug conjugates of the group of antibody-drug conjugates is selected from integers or decimals of 1 to 16, preferably 1 to 10.

[0142] In some embodiments, the average ratio of drug to antibody (average DAR) in the group of antibody-drug conjugates is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5, or 7.5 to 8.5.

[0143] In some embodiments, the average ratio of drug to antibody (average DAR) in the group of antibody-drug conjugates is selected from about 2.0, 4.0, 6.0, or 8.0.

[0144] In some embodiments, the average ratio of drug to antibody (average DAR) in the antibody-drug conjugates of the group of antibody-drug conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0145] In some embodiments, the group of antibody-drug conjugates contains ADCs having a DAR distribution of 1 to 8, such as 1.5, 2, 4, 6, and 8 (i.e., drug-loading types of 1.5, 2, 4, 6, and 8). It should be noted that degradation products may be generated so that DARs of 1, 3, 5, and 7 may also be included in the mixture. Further, the group of antibody-drug conjugates can also have an average DAR greater than 8. The antibody-drug conjugate is generated by reducing the interchain disulfide and then coupling. In some embodiments, the antibody-drug conjugate includes both the antibody-drug conjugate with a DAR of 4 or less (i.e., the drug-loading type is 4 or less) and the antibody-drug conjugate with a DAR of 6 or more (i.e., the drug-loading type is 6 or more).

[0146] In a tenth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition, which comprises an antibody or an antigen-binding fragment thereof according to the first aspect, a multi-specific antibody according to the second aspect, an antibody-drug conjugate according to the seventh aspect or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof or a pharmaceutically acceptable solvate thereof, a nucleic acid according to the third aspect, an immune complex according to the fifth aspect or a group of antibody-drug conjugates according to the ninth aspect, and optionally one or more pharmaceutical additives, such as a buffer, a pharmaceutical carrier known in the art, and a pharmaceutical excipient.

[0147] In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody or an antigen-binding fragment thereof according to the first aspect, a multi-specific antibody according to the second aspect, an antibody-drug conjugate according to the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof or a pharmaceutically acceptable solvate thereof, a nucleic acid according to the third aspect, an immune complex according to the fifth aspect or a group of antibody-drug conjugates according to the ninth aspect.

[0148] In certain embodiments, the pharmaceutical composition comprises the above antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof, and a pharmaceutical additive.

[0149] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the antibody-drug conjugate group of the ninth aspect and a pharmaceutical additive.

[0150] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the anti-c-MET antibody of the first aspect of the present disclosure or an antigen-binding fragment thereof, and a pharmaceutical additive.

[0151] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises the isolated nucleic acid molecule or carrier described above.

[0152] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the multispecific antibody of the second aspect of the present disclosure and a pharmaceutical additive.

[0153] In some embodiments, the ratio (average DAR) of the drug to the antibody in the pharmaceutical composition or the antibody-drug conjugate group is selected from an integer or decimal number from 1 to 10.

[0154] In some embodiments, the ratio (average DAR) of the drug to the antibody in the pharmaceutical composition or the antibody-drug conjugate group is selected from 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5, and 7.5 to 8.5.

[0155] In some embodiments, the DAR in the pharmaceutical composition or the antibody-drug conjugate group is selected from 2±0.5, 4±0.5, 5±0.5, 6±0.5, 7±0.5, 8±0.5.

[0156] In some embodiments, the ratio (average DAR) of the drug to the antibody in the pharmaceutical composition or the antibody-drug conjugate group is selected from about 2.0, 4.0, 6.0, or 8.0.

[0157] In some embodiments, the ratio of the drug to the antibody in the drug composition or the group of antibody-drug conjugates (average DAR) is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9 or 9.7.

[0158] In some embodiments, the drug composition comprises an antibody of the first aspect or an antigen-binding fragment thereof, a multispecific antibody of the second aspect, or a drug conjugate of the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof or a pharmaceutically acceptable solvate and buffer thereof.

[0159] In some embodiments, the drug composition comprises a drug conjugate of the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof or a pharmaceutically acceptable solvate and buffer thereof.

[0160] In some preferred embodiments, the buffer is selected from histidine buffer and phosphate buffer. In some preferred embodiments, the buffer is selected from histidine buffer. In some preferred embodiments, the buffer is selected from 20 mM histidine buffer.

[0161] In the eleventh aspect, there is provided the use of an antibody or an antigen-binding fragment thereof according to the present disclosure in the preparation of a kit, wherein the kit is used for detecting the presence or level of c-MET in a sample. In another aspect, the present disclosure provides a diagnostic kit or a therapeutic kit, which kit comprises one or more of the antibody or an antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, antibody-drug conjugate, group of antibody-drug conjugates or drug composition according to the present disclosure. Optionally, the diagnostic kit or the therapeutic kit further comprises instructions for use. In some embodiments, the kit is applicable to the diagnostic or detection method according to the present disclosure. In some embodiments, the kit is applicable to the therapeutic method according to the present disclosure.

[0162] In a 12th aspect, the present disclosure provides the use of a substance of the present disclosure (including the above antibody-drug conjugate composition or the above drug composition) in the preparation of a drug for treating and / or preventing a disease associated with abnormal cell activity (e.g., a cancer disease). In some embodiments, the antibody-drug conjugate composition or the above drug composition is an effective amount such as a therapeutically effective amount.

[0163] In some embodiments, the present disclosure provides the use of an anti-C-MET antibody or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, or a multispecific antibody of the present disclosure in the preparation of a drug, and the drug is used to regulate (inhibit or block) the activity of C-MET.

[0164] In some embodiments, the present disclosure provides the use of an anti-C-MET antibody or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell antibody-drug conjugate or a multispecific antibody of the present disclosure in the preparation of a drug, and the drug is used for the treatment or prevention of a disease associated with the activity of C-MET or a disease associated with a target of C-MET.

[0165] In some embodiments, the present disclosure provides the use of an anti-C-MET antibody or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, an antibody-drug conjugate, or a multispecific antibody of the present disclosure in the preparation of a drug, and the drug is used for the treatment or prevention of a tumor associated with the activity of C-MET.

[0166] In some embodiments, the present disclosure provides the use of a substance of the present disclosure in the preparation of a drug, and the drug is used for the treatment or prevention of a disease associated with the activity of C-MET or a disease associated with a target of C-MET. The substance of the present disclosure is selected from the above antibody-drug conjugate of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, the anti-C-MET antibody or its antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid described in the third aspect, the vector described in the fourth aspect, the vector described in the sixth aspect, the immune complex of the seventh aspect or the group of antibody-drug conjugates of the ninth aspect, or the drug composition of the tenth aspect.

[0167] In some embodiments, provided is the use in the preparation of a drug of the antibody or antigen-binding fragment thereof of the first aspect, the multi-specific antibody of the second aspect, the antibody-drug conjugate of the seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers or its pharmaceutically acceptable solvates, the group of antibody-drug conjugates of the ninth aspect, or the drug of the pharmaceutical composition of the tenth aspect of the present disclosure, wherein the drug is used for the treatment or prevention of a disease associated with the activity of C-MET or a disease associated with a target of C-MET.

[0168] In a thirteenth aspect, the present disclosure provides a method of using the antibody or antigen-binding fragment thereof of the first aspect, the multi-specific antibody of the second aspect, the antibody-drug conjugate of the seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers or its pharmaceutically acceptable solvates, the nucleic acid according to the third aspect, the vector according to the fourth aspect, the immune complex of the fifth aspect or the group of antibody-drug conjugates of the ninth aspect, or the pharmaceutical composition of the tenth aspect, for the treatment and / or prevention of a disease associated with abnormal cell activity (e.g., a tumor).

[0169] In the above twelfth and thirteenth aspects, the disease associated with the activity of C-MET or the disease associated with a target of C-MET or the disease associated with abnormal cell activity includes tumors such as cancer. The cancer may be early-stage, middle-stage, late-stage, or metastatic cancer. In some embodiments, the cancer may be a solid tumor or a hematological tumor.

[0170] In one embodiment, the tumor means that in the tumor tissue or tumor cells of an individual, for example, compared to the adjacent normal tissue or normal cells (e.g., normal cells in the tissue) of the individual, or the same tissue or cells therein in a healthy individual, the protein level (e.g., expression) of c-MET is increased, or the nucleic acid level of c-MET is increased.

[0171] The tumor is selected from, but not limited to, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer or lung adenocarcinoma), colon cancer (e.g., human colon adenocarcinoma), rectal cancer, gastric cancer, colorectal cancer (e.g., colorectal adenocarcinoma).

[0172] In the 12th and 13th aspects above, the antibody or antigen-binding fragment thereof of the 1st aspect, the multispecific antibody of the 2nd aspect, the antibody-drug conjugate of the 7th aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, the nucleic acid described in the 3rd aspect, the vector described in the 4th aspect, the immune complex of the 5th aspect or the group of antibody-drug conjugates of the 9th aspect, or the pharmaceutical composition of the 10th aspect can also be combined with other treatment methods or therapeutic agents for treating related diseases or for related uses.

[0173] In some embodiments, the treatment method is surgical treatment or radiotherapy.

[0174] In the 14th aspect, the present disclosure further provides a drug combination or a drug combination product, and the drug combination or the drug combination product includes an anti-c-MET antibody or a fragment thereof (preferably an antigen-binding fragment) of the present invention, or an antibody-drug conjugate thereof, and one or more other therapeutic agents.

[0175] Another object of the present invention is to provide a medicine case in the form of a set including the drug combination of the present invention, preferably the medicine case is in the form of a drug administration unit. Therefore, the administration unit can be provided according to a dosing plan or a drug administration interval.

[0176] In one embodiment, the medicine case in the form of a set of the present invention contains, within the same package, - a first container containing a pharmaceutical composition containing an anti-c-MET antibody or a fragment thereof, - a second container containing a pharmaceutical composition containing other therapeutic agents.

[0177] In a 15th aspect, the present disclosure further provides the use of the antibody or antigen-binding fragment thereof of the first aspect, or the multispecific antibody of the second aspect, in the preparation of an antibody-drug conjugate.

[0178] In some embodiments, the antibody-drug conjugate is selected from the antibody-drug conjugate of the 7th aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate.

[0179] The immunoglobulin molecules of the present disclosure may be of any type of immunoglobulin (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Preferably, the antibody of the present disclosure comprises, or consists of, the VH domain, VH CDR (mainly denoted as HCDR herein), VL domain, or VL CDR (mainly denoted as LCDR herein) of any one amino acid sequence or its fragment or variant described in the sequence or specific information table.

[0180] Preferably, the antibody of the present disclosure comprises, or consists of, the VH domain, VH CDR (mainly denoted as HCDR herein), VL domain, or VL CDR (mainly denoted as LCDR herein) of any one amino acid sequence or its fragment or variant described in the sequence or specific information table.

[0181] In the present disclosure, the term "monoclonal antibody" means that the antibody is derived from a substantially homogeneous population of antibodies, i.e., each antibody constituting the cluster is identical, except for the possibility of a few natural mutations. The modifier "monoclonal" here indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring preparation by a particular method.

[0182] In some embodiments of the present disclosure, the monoclonal antibody further particularly includes chimeric antibodies, that is, as long as it has the required biological activity, a part of the heavy chain and / or light chain is the same or homologous to an antibody of a certain species, a certain class or a certain subclass, and other parts are the same or homologous to an antibody of another species, another class or another subclass. Chimeric antibodies that can be used in the present disclosure include primatized antibodies containing variable region antigen-binding sequences derived from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0183] The term "antigen-binding fragment" refers to a part of an antibody, preferably the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’) 2 , Fd, Fv, dAb and complementarity-determining region fragments, diabodies, linear antibodies and single-chain antibody molecules. The term "antigen-binding fragment" as used herein represents a fragment of a part of an antibody having antigen-binding activity, where the fragment has all or part of the function of the antibody, including single-chain Fv (scFv), Fab, Fab’, F(ab’) 2 , disulfide-bonded Fv (sdFv), Fv, di-scFv, etc., but not limited thereto. This term also includes Fab’, which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab’)2 under reducing conditions. However, as long as the fragment has an affinity for binding to an antigen, this term is not limited to these molecules. Furthermore, these functional fragments include not only fragments obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also proteins produced in an appropriate host cell using a genetically modified antibody gene.

[0184] The term "Fab’" as used herein means a monovalent fragment of the variable region of an antibody obtained by treating F(ab’) 2 under the above-mentioned reducing conditions. However, the Fab’ of the present disclosure also includes Fab’ produced using a genetically modified antibody gene.

[0185] As used herein, the term "scFv" means a single polypeptide chain comprising VL and VH domains, where said VL and VH are linked via a linker or directly linked (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pages 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH, or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. Optionally, a disulfide bond can also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" means an antibody fragment formed by the binding of two scFvs.

[0186] The term "variable region" or "variable domain" or "variable domain" means the domain related to the antibody-binding antigen in the heavy chain and / or light chain of an antibody. Native IgG antibody VH and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). Here, the term "complementarity-determining region" or "CDR" means the region mainly promoting the binding to an antigen within the variable domain, and "framework" or "FR" means the variable domain residues other than the CDR residues within the variable domain. VH contains three CDR regions of HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions of LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0187] The boundaries of the amino acid sequences of CDRs can be determined by various known definition schemes. For example, the "Kabat" definition scheme rules (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" definition scheme, the "ABM" definition scheme, the "contact" definition scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) definition scheme (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55 - 77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc. The correspondence relationships between various definition schemes are well known to those skilled in the art and are exemplary and are shown as follows.

Table 11

[0188] As used herein, even if the antibody or antigen-binding fragment thereof contains a variant, amino acid substitution, deletion or addition, it still has the activity of binding to the antigen.

[0189] The term "binding molecule" means any molecule that can specifically bind to a target, such as an antibody or an antigen-binding fragment thereof or a fusion protein. The term "multispecific binding molecule" means a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule, i.e., the molecule includes at least a first target-binding region and a second target-binding region, where the first target-binding region binds to one target or antigen, and the second target-binding region binds to another antigen or target. The multispecific binding molecules according to the present invention also include multispecific molecules containing multiple target-binding regions such as trispecific binding molecules. In some embodiments, the multispecific binding molecules of the present invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies.

[0190] The term "bispecific antibody", also referred to as a "bifunctional antibody complex", means a complex formed by a first antibody (fragment) and a second antibody (fragment) via a coupling arm, and since the activity of each antibody is retained in the complex, it has bifunctionality and bispecificity. In one embodiment, the present specification provides such a bispecific antibody, which has binding specificity for C-Met and binding specificity for a second antigen.

[0191] The term "multispecific antibody" includes, for example, bispecific antibodies, trispecific antibodies and tetravalent antibodies, the former being antibodies having three different antigen-binding specificities, and the latter being antibodies having four different antigen-binding specificities. In some embodiments, the multispecific antibody has binding specificity for c-Met and one or more binding specificities for other antigens.

[0192] The term "intact antibody" or "full-length antibody" means an antibody that includes an antigen-binding variable region and a light-chain constant region (CL), and heavy-chain constant regions (CH1, CH2, and CH3). The constant regions may be of a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions. In the present disclosure, the "humanized" form of a non-human (e.g., mouse) antibody means a chimeric antibody containing a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies have the hypervariable region residues of a human recipient immunoglobulin replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Additionally, a humanized antibody can contain residues not present in the recipient antibody or donor antibody. These modifications are intended to further optimize the performance of the antibody. A humanized antibody generally includes at least one, usually two, variable regions, with all or substantially all of the hypervariable loops corresponding to the non-human immunoglobulin and the FR being fully or substantially fully of human immunoglobulin sequence. A humanized antibody can further include at least a portion of an immunoglobulin constant region (Fc, usually a human immunoglobulin Fc).

[0193] Intact antibodies can be classified into different "classes" based on the amino acid sequence of the heavy-chain constant region. The five major classes are IgA, IgD, IgE, IgG, and IgM, and some of these can also be classified into different "subclasses" (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. The heavy-chain constant regions of the different classes of antibodies are designated α, β, ε, γ, and μ, respectively. The different subunit structures and three-dimensional configurations of immunoglobulins are well known in the art.

[0194] As used herein, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure can be determined according to various definition schemes known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present disclosure are preferably determined by the Kabat, Chothia, AbM or IMGT definition schemes.

[0195] As used herein, the term "framework residue region" or "FR residue" means those amino acid residues in the antibody variable region other than the CDR residues defined above.

[0196] The 20 conventional amino acids described herein are described according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably with each other. And in the present disclosure, amino acids are usually represented by the one-letter and three-letter abbreviations well-known in the art. For example, alanine can be represented by A or Ala, arginine can be represented by R or Arg, glycine can be represented by G or Gly, and glutamine can be represented by Q or Gln.

[0197] As used herein, the term "immunoconjugate" means herein that an effector molecule is linked to an antibody or an antigen-binding fragment thereof via a linker, whereby the antibody or an antigen-binding fragment thereof can be used as a vector to target and deliver the effector molecule to a target site. The term "effector molecule" means an active moiety that binds to an antibody or antibody fragment of the invention and can include any moiety used to attach to the antibody or antibody fragment. In some embodiments, the effector molecule may be a drug such as a small molecule drug, DNA, RNA, enzyme or polypeptide. In some embodiments, the immunoconjugate includes an antibody-drug conjugate (ADC). Effector molecules or active moieties suitable for binding to an antibody include, for example, anti-tumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions and any combination thereof. In some embodiments, the immunoconjugate of the invention is an antibody-drug conjugate, i.e., an ADC.

[0198] As used herein, the term "immunomodulator" refers to a natural or synthetic activator or drug that inhibits or modulates (e.g., activates) an immune response. The immune response may be a humoral response or a cellular response. Immunomodulators include immunosuppressive agents. In some embodiments, the immunomodulator of the invention includes an immune checkpoint inhibitor or an immune checkpoint agonist.

[0199] As used herein, the term "prevention" means a method performed in a subject's body to prevent or delay the occurrence of a disease, disorder or symptom (e.g., tumor and infection). As used herein, the term "treatment" means a method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the scope of the disease, stabilization of the disease state (i.e., no worsening), delay or reduction in the progression of the disease, improvement or alleviation of the disease state, alleviation (partial or complete) of symptoms, whether detectable or undetectable. Further, "treatment" also means extending the survival period compared to the expected survival period (if not treated).

[0200] As used herein, the term "subject" means a mammal, such as a non-human primate mammal or a primate mammal such as a human. In certain embodiments, the subject (e.g., a human) suffers from a tumor and an infection, or has a risk of suffering from the above diseases.

[0201] As used herein, the term "effective amount" means an amount sufficient to obtain or at least partially obtain a desired effect. For example, an effective amount for preventing a disease (e.g., tumor and infection) means an amount sufficient to prevent, inhibit or delay the occurrence of the disease (e.g., tumor and infection), and an effective amount for treating a disease means an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. Measuring such an effective amount is well within the ability of one of ordinary skill in the art. For example, for an amount effective for therapeutic use, it varies depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the general state of the patient such as age, weight and gender, the method of drug administration, and other treatments administered simultaneously.

[0202] The term "pharmaceutical additive" means a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, vector or stabilizer, etc., administered together with an active substance.

[0203] The term "pharmaceutical composition" means a composition that enables the biologically active effective form of the active ingredient contained therein to exist and that does not contain other ingredients having unacceptable toxicity to the subject to whom the composition is administered.

[0204] The term "drug combination or combination product" means a non-fixed combination product or a fixed combination product, including, but not limited to, a medicine case / kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibody of the present invention and (ii) other therapeutic agents) are administered to a patient simultaneously as separate entities, without a specific time limit, or sequentially at the same or different time intervals, and such administration provides a prophylactically or therapeutically effective level of two or more active agents to the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosages and / or time intervals of two or more active agents are selected such that the combined use of each component can produce an effect greater than the effect achievable by any one component alone in the treatment of a disease or disorder. Each component may be in a separate dosage form, which may be the same or different.

[0205] The term "combination therapy" means administering two or more therapeutic agents or therapies for treating the diseases described herein. Such administration includes, for example, co-administering the therapeutic agents substantially simultaneously in a single capsule having a fixed ratio of active ingredients. Or such administration includes co-administering each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dosage before administration. Further, such administration further includes administering each type of therapeutic agent either substantially simultaneously or sequentially at different times. In any case, the treatment plan provides the beneficial effect of the drug combination in the treatment of the disorders or conditions described herein.

[0206] As used herein, the term "label" means a compound or composition that is directly or indirectly bound or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates the detection of the reagent to which it is bound or fused. The label itself may be detectable (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, it can catalyze a detectable chemical change in a substrate compound or composition. This term is intended to encompass directly labeling a probe or antibody by binding a detectable substance to the probe or antibody (i.e., physically binding it), and indirectly labeling a probe or antibody by reacting it with another directly labeled reagent.

[0207] An "isolated" antibody or molecule is one that is separated from the components of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0208] The "percent identity (%)" of an amino acid sequence means the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown herein when the candidate sequence is aligned with the specific amino acid sequence shown herein and, if necessary, gaps are introduced to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention, and the variants have a significant degree of identity to the antibody molecules and their sequences specifically disclosed herein. For example, the identity is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more. The variants can include conservative changes.

[0209] The term "about," when used in combination with a numerical value, means a numerical range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0210] As used herein, the term "and / or" means any one or more of the options.

[0211] As used herein, the term "comprising" or "including" means including the recited element, integer or step, but not meaning to exclude any other element, integer or step. As used herein, the term "comprising" or "including", when used herein, includes combinations of the recited elements, integers or steps, unless otherwise indicated. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to include an antibody variable region consisting of that particular sequence.

[0212] The term "pharmaceutically acceptable" means that a molecular entity, molecular fragment or composition does not produce adverse effects, allergic reactions or other harmful reactions when appropriately administered to an animal or a human. Specific examples of substances that can be pharmaceutically acceptable vectors or components thereof include saccharides (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (e.g., propylene glycol), alginic acid, etc.

[0213] The term "drug-to-antibody ratio" or "DAR" means the ratio of the amount of drug moiety (D) bound to the antibody moiety (Ab) described herein to the antibody moiety. The DAR of an ADC can be in the range of 1 to 20, but higher loadings are also possible depending on the number of binding sites on the antibody. The term DAR can be used when referring to the amount of drug carried on a single antibody or alternatively when referring to the average or mean DAR of a set of ADCs. The DAR may also be calculated as the average DAR of the population of molecules in the product, i.e., the overall ratio (molar ratio) of the drug moiety (D) bound to the Ab moiety described herein in the product measured by a measurement method (e.g., conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), and this DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the antibody-drug conjugate of the present disclosure is from 1.0 to 20.0, such as from 1.0 to 18.0, from 1.0 to 16.0, from 2.0 to 14.0, from 3.0 to 12.0, from 4.0 to 10.0, from 5.0 to 9.0, from 6.0 to 8.0, from 1.0 to 8.0, from 2.0 to 6.0, and for example, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 12.0, and 16.0, with the range being between two of these values as endpoints.

[0214] In the process of determining the DAR value by mass spectrometry, the antibody is decomposed into separated heavy and light chains. DAR1 represents a complex containing a light or heavy chain bound to one toxin molecule, DAR2 represents a complex containing a light or heavy chain bound to two toxin molecules, and DAR3 represents a complex containing a light or heavy chain bound to three toxin molecules.

[0215] The term "drug" refers to a chemical substance that can change or determine the physiological functions or pathological states of the body and can be used for the prevention, diagnosis, and treatment of diseases, and particularly includes substances that suppress or prevent the functions of cells and / or cause cell death or destruction. Drugs include cytotoxic agents, particularly small molecule cytotoxic agents. There is no strict boundary between drugs and poisons. A poison means a chemical substance that has a toxic effect on the human body in small amounts and impairs human health, and any overdose of a drug may cause a toxic reaction.

[0216] A cytotoxic agent is a substance that suppresses or prevents the functions of cells and / or causes cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, they may cause apoptosis of normal cells and severe side effects while killing tumor cells. Cytotoxic agents include toxins such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, radioisotopes (e.g., At 211 , I 13 1, I 125 , Y 90 , Re 186 , Re 188 , Sm 15 , Bi 212 , P 32and radioisotopes of Lu), chemotherapeutic agents, antibiotics, and nucleolytic enzymes. It also includes DNA topoisomerase inhibitors (e.g., camptothecin, DXD, camptothecin with modified substituents or DXD with modified substituents, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, and other camptothecin-based bioactive molecules), or microtubulin inhibitors (e.g., MMAF-type microtubulin inhibitors, MMAE-type microtubulin inhibitors), but is not limited thereto.

[0217] To avoid ambiguity, the "drug" that can be a component of the ADC does not only mean the "drug" approved by the medical regulatory authorities, but also includes any compound with potential therapeutic bioactivity in clinical, or research and development and academic research. It should also be understood that it has a different meaning from the "medicine" in the "use in the preparation of medicines". To bind to the linker, it is necessary to functionalize or derivatize the drug molecule, and it should be understood that the compound thus obtained is also included in the pharmaceutical category of the present disclosure.

[0218] In the present disclosure, the "active drug unit" refers to the part other than the antibody and the linker of the present disclosure in an antibody-drug conjugate (or, referred to as an antibody-drug conjugate, ADC), which is derived from the drug defined above. For convenience, the "active drug unit" in the ADC of the present disclosure can be directly called by the name of the above drug, which is understood by those skilled in the art.

[0219] As used herein, the term "pharmaceutically acceptable salt" means a salt that maintains the biological effects and properties of the antibody-drug conjugate or drug-linker conjugate of the present disclosure, and the salt is not biologically or otherwise undesirable. The conjugates of the present disclosure (including antibody-drug conjugates and drug-linker conjugates) can exist in the form of pharmaceutically acceptable salts, including their acid addition salts and base addition salts. In the present disclosure, pharmaceutically acceptable acid addition salts represent salts formed from the conjugates of the present disclosure and organic or inorganic acids, and the organic or inorganic acids include, but are not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable base addition salts represent salts formed from the conjugates of the present disclosure and organic or inorganic bases, and include, but are not limited to, alkali metal salts such as lithium, sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, organic base salts, and organic bases such as ammonium salts formed from organic bases containing an N group.

[0220] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0221] As used herein, the term "stereoisomer" means an isomer formed by at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers can be produced. Certain individual molecules can exist as geometric isomers (cis / trans). Similarly, the compounds of the present disclosure can exist as mixtures of two or more structurally different forms (usually called tautomers) in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, and imine-enamine tautomers. The scope of the present disclosure encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0222] In the present disclosure, solid lines (--), solid wedges, or dashed wedges can be used to indicate carbon-carbon bonds of the compounds of the present disclosure. The use of a solid line to depict a bond attached to an asymmetric carbon atom indicates that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at this carbon atom are included. The use of a solid wedge or a dashed wedge to depict a bond attached to an asymmetric carbon atom indicates the presence of the indicated stereoisomer. Unless otherwise specified, it is intended that the compounds of the present disclosure may exist in the form of stereoisomers (including cis and trans isomers, optical isomers (e.g., R enantiomers and S enantiomers), diastereoisomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the present disclosure can exhibit one or more isomerization phenomena and consist of mixtures thereof (e.g., racemic mixtures and diastereoisomer pairs).

[0223] The present disclosure further includes pharmaceutically acceptable isotopic compounds that are identical to the compounds of the present disclosure, except that one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the preponderant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of the present disclosure include isotopes of hydrogen (e.g., 2 H, 3 H), isotopes of carbon (e.g., 11 C, 13 C, and 14 C), isotopes of chlorine (e.g., 36 Cl), isotopes of fluorine (e.g., 18 F), isotopes of iodine (e.g., 123 I and 125 I), isotopes of nitrogen (e.g., 13 N and 15 N), isotopes of oxygen (e.g., 15 O, 17 O, and 18 O), isotopes of phosphorus (e.g., 32 P), and isotopes of sulfur (e.g., 35 S) (including, but not limited to, these).

[0224] The compounds of the present disclosure can exist in the form of solvates (preferably hydrates), where the compounds of the present disclosure include a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the crystal lattice of the compound. The amount of the polar solvent, particularly water, can be present in a stoichiometric or non-stoichiometric ratio.

[0225] Also within the scope of the present disclosure are metabolites of the compounds of the present disclosure, i.e., substances formed in the body upon administration of the compounds of the present disclosure. Such products can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, de-esterification, enzymatic degradation, etc. of the administered compound. Accordingly, the present disclosure includes metabolites of the compounds of the present disclosure and compounds produced by a method of contacting a mammal with a compound of the present disclosure for a time sufficient to produce its metabolites.

[0226] Within its scope, the present disclosure further includes prodrugs of the compounds of the present disclosure. Generally, such prodrugs are functional group derivatives of said compounds, which are readily convertible in vivo to the desired therapeutically active compounds. Thus, in these cases, the term "administration" as used in the treatment methods of the present disclosure should include the treatment of various diseases or disorders with one or more prodrug forms of the compounds for which protection is claimed, provided that the prodrug forms are converted in vivo to the above compounds after being administered to an individual. For example, "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985 describes general methods for selecting and preparing suitable prodrug derivatives.

[0227] In the present disclosure, the pharmaceutical additive means excipients and additives used in the manufacture of pharmaceuticals and the preparation of prescriptions, and refers to substances contained in pharmaceutical preparations that are rationally evaluated from the perspective of safety in addition to the active ingredient. Pharmaceutical additives or excipients have important functions such as solubilization, cosolvency, sustained release, and controlled release, in addition to shaping, functioning as a vector, and improving stability, and are important components that can affect the quality, safety, and efficacy of pharmaceuticals. Depending on their origin, they can be classified into natural products, semi-synthetic products, or fully synthetic products. Depending on their functions and uses, they can be classified into solvents, propellants, solubilizing agents, cosolvents, emulsifiers, coloring agents, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, taste improvers, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, moisturizing agents, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. Depending on the administration route, they can be classified into oral administration, injection, mucosa, transdermal or topical administration, nasal or oral inhalation administration, ophthalmic administration, etc. The same pharmaceutical additive or excipient can be used in drug preparations with different administration routes and has different functions and uses. For the use and applications of pharmaceutical additives, reference should also be made to "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0228] The pharmaceutical composition can be prepared into various appropriate dosage forms according to the administration route. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powders, sprays, etc. Here, the pharmaceutical composition or appropriate dosage form can contain 0.01 mg to 1000 mg of the compound (including the conjugate) of the present disclosure or a pharmaceutically acceptable salt thereof, appropriately 0.1 mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg, particularly preferably 1 to 250 mg. There may be cases where the amount exceeds the above range.

[0229] The pharmaceutical composition can be administered in the form of an injection, including injection solutions, sterile powders for injection, and concentrated injection solutions. Here, usable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Also, sterilized non-volatile oils can be used as solvents or suspension media such as monoglycerides or diglycerides. The pharmaceutical composition can be administered in the form of an infusion.

[0230] Regarding "L is bound to the antibody via a sulfur atom" in the present disclosure, the sulfur atom is derived from the sulfhydryl contained in the antibody itself after opening the disulfide bond (for example, reduction of the disulfide bond by a reducing agent TCEP can open the disulfide bond to generate sulfhydryl -SH). That is, it can be understood by those skilled in the art that the -S- between L and Ab is not an additional foreign sulfur atom.

[0231] As used herein, the term "comprising" or "including" means including the recited element, integer, or step, but does not mean excluding any other element, integer, or step. In this specification, when the term "comprising" or "including" is used, unless otherwise specified, it also includes the case consisting of the recited element, integer, or step. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to include an antibody variable region consisting of the specific sequence.

[0232] In the present disclosure, the term "linking group" or "linker" means a fragment that links an active drug unit (drug molecule) to an antibody moiety. In this regard, the linker has a functional group capable of forming a bond with a functional group of an antibody or an antigen-binding fragment thereof (i.e., a linker precursor) before binding to the antibody or its antigen-binding fragment.

[0233] In the present disclosure, the term "antibody-drug conjugate" or "ADC" means a substance obtained by binding an active drug unit (drug molecule) to an antibody or an antigen-binding fragment thereof. In some embodiments of the present disclosure, the active drug unit and the targeting moiety are linked via a linker. The linker can be cleaved under a specific environment (e.g., a low pH environment inside a cell) or a specific action (e.g., the action of lysosomal protease), thereby isolating a fragment of a bioactive compound (e.g., a c-Myc proteolytic agent) from the target moiety or the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker includes a cleavable or non-cleavable unit such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the active drug unit is directly linked to the target moiety or the antibody or its antigen-binding fragment via a covalent bond, and the covalent bond can be cleaved under a specific situation or action, thereby partially isolating the active drug unit from the antibody or its antigen-binding fragment.

[0234] In the present disclosure, the term "alkyl group" means a straight-chain or branched-chain fully saturated hydrocarbon group that may be optionally substituted, preferably a C 1 ~C 10 alkyl group, more preferably a C 1 ~C 8 alkyl group, a C 1 ~C 6 alkyl group, or a C 1 ~C 4 alkyl group. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-butyl.

[0235] In the present disclosure, the term "aryl" means a monocyclic (e.g., phenyl) or fused ring (e.g., naphthyl, anthracenyl, phenanthrenyl, fluorenyl, etc.) optionally substituted C 6 ~C 16 aromatic hydrocarbon group, preferably a C 6 ~C 10 aromatic hydrocarbon group.

[0236] In the present disclosure, the term "heteroaryl group" means a 5- to 16-membered aromatic group, preferably a 5- to 10-membered aromatic group, more preferably a 5- to 6-membered aromatic group, optionally substituted and containing one or more (e.g., 1, 2, 3, or 4) heteroatoms selected from N, O, S, or P. Examples of heteroaryl groups include imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyrrolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indole, azaindole (e.g., 7-azaindole), benzimidazole, benzopyrazole, benzofuranyl, benzothienyl, benzothiazole, dibenzofuranyl, dibenzothienyl, quinolinyl, isoquinolinyl, naphthyridinyl, carbazolyl, azacarbazolyl (e.g., 1-azacarbazolyl, 2-azacarbazolyl, 1,8-diazacarbazolyl), indolazinyl, azaindolazinyl, phenoxazinyl, phenothiazinyl, etc.

[0237] As used herein, the term "3- to 6-membered cycloalkyl group" or "C 3~6 cycloalkyl group" means a saturated cyclic alkyl group containing 3 to 6 carbon atoms, and includes cyclopropane (i.e., cyclopropyl group), azetidinyl (i.e., cyclopentyl group), cyclopentanyl (i.e., cyclopentyl group), cyclohexyl group.

[0238] As used herein, the term "5- or 6-membered heterocyclic ring" means a ring containing 5 to 6 ring atoms, at least one (e.g., 1, 2, or 3) of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom, and includes, but is not limited to, rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, and tetrahydropyran.

[0239] As used herein, the term "5- or 6-membered heterocyclyl" means a cyclic group containing 5 to 6 ring atoms, at least one (e.g., 1, 2, or 3) of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom, and includes, but is not limited to, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, etc.

[0240] The term "antibody-drug conjugate group" means a mixture consisting of one or a group of the disclosed antibody-drug conjugates, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates, where the q of the antibody-drug conjugates may be the same or different. It may also be referred to as an "antibody-drug conjugate mixture".

[0241] Advantageous Effects of the Invention In some embodiments, the c-MET targeting antibody or antigen-binding fragment thereof developed in the present disclosure binds to c-Met with high affinity, has high affinity for human and monkey cMet, and at the same time has good binding specificity and does not bind to rat and mouse cMet.

[0242] In some embodiments, the c-MET targeting antibody or antigen-binding fragment thereof developed in the present disclosure has an antagonistic effect, not an agonist effect, and can antagonize HGF-induced c-Met and protein kinase B (PKB) phosphorylation antibodies.

[0243] In some embodiments, compared to control antibodies such as ABT700, the antibodies targeting c-MET or antigen-binding fragments thereof developed according to the present disclosure have efficient endocytosis activity, higher affinity, different binding epitopes, and compete with the binding activity of HGF ligand to c-Met.

[0244] In some embodiments, the anti-c-MET antibodies provided by the present disclosure have a very high degree of humanization and / or thermal stability, whereby they can be used for drug creation without inducing an immunogenic response and can be safely administered to human subjects.

[0245] Therefore, in some embodiments, the antibodies or antigen-binding fragments thereof targeting c-MET of the present disclosure exhibit specificity, reduced toxicity, stability, and enhanced physical and functional properties compared to known therapeutic agents.

[0246] At the same time, while the development of c-MET signal blockade inhibitors only benefits a portion of the total number of tumor patients expressing c-MET, the c-MET ADCs of the present disclosure overcome some limitations of signal blockade c-MET inhibitors and can benefit more tumor patients with low c-MET expression.

[0247] In some embodiments, the antibody-drug conjugates (ADCs) of the present disclosure achieve a screening verification that combines tumor microenvironment enrichment, linker-specific in vivo enzyme cleavage characteristics and binding mode with the target moiety, and in vitro and in vivo efficacy in large quantities, resulting in a novel antibody bioactive molecule conjugate. Using the conjugate obtained by the above method, various surprising technical effects can be achieved as follows. In some embodiments, the conjugate obtained according to the above method has better solubility and excellent chemical stability. For example, since the reversible Michael addition reaction caused by the maleimide binding mode in conventional ADCs does not occur, a high DAR value can be obtained. In some embodiments, the DAR value of the conjugate can reach 6-8 or even higher. It has a very high binding efficiency, and in some embodiments, the binding efficiency reaches 90% or more. Thus, in one embodiment, through extensive research, the present disclosure has discovered an ADC that has high plasma stability but is cleavable in the tumor microenvironment (both inside and outside tumor cells), enabling release both inside tumor cells and in tumor tissue, maximizing the delivery of the ADC to tumor tissue and tumor cells, thereby enabling its effectiveness to be exerted in the treatment of tumors, and thus excellent anti-tumor effects can be exerted even in tumors with low antigen expression or no antigen expression. The ADCs of the present disclosure showed obvious tumor inhibitory activity against tumor cells with different c-MET expression levels.

[0248] In some embodiments, after the ADCs of the present disclosure were incubated at 37 °C for 504 h, the release rates of the toxins in PBS solution, cynomolgus monkey plasma, and human plasma were each <0.6%, having excellent plasma stability and circulation stability. The conjugate (ADC) obtained according to the above method improves the exposure amount of the whole ADC molecule in a relatively acidic tumor environment by adjusting the physical and chemical properties of the linker and the whole ADC molecule. As a result, the ADC has better tumor tissue targeting, that is, the ability to concentrate in the tumor microenvironment, increases the ratio of the concentration of the bioactive molecule in the tumor to that in the blood, and reduces the mechanism-related toxicity of the ADC molecule (the toxicity caused by the ADC binding to the cell surface antigen of non-tumor tissue and undergoing endocytosis, or "on-target toxicity"), thereby having a higher therapeutic index. In some embodiments, the conjugate obtained according to the above method has high stability in in vivo circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target" toxicity caused by the shedding of toxins in non-target tissues. In some embodiments, the bioactive molecule of the conjugate has higher anti-tumor cell activity and thus has an excellent by-stander effect. The ADC can more effectively kill tumor cells with high antigen expression and tumor cells with low or no antigen expression in tumor tissue. In some embodiments, the antibody-drug conjugate of the present disclosure can utilize the extracellular cleavage ability of its linker in the tumor microenvironment to form an antibody-binding drug with an antibody that does not have the ability of cellular endocytosis, and such an antibody-binding drug still has high antitumor activity. In some embodiments, the antibody-drug conjugate of the present disclosure can utilize the extracellular cleavage ability of its linker in the tumor microenvironment and its concentration ability in the tumor microenvironment to form an antibody-binding drug together with an antibody that does not have the ability of cellular endocytosis and an antibody that does not have the ability to bind to extracellular tumor antigens, and such an antibody-binding agent still has high antitumor activity. In summary, the ADC of the present disclosure has significant clinical value.

Brief Description of the Drawings

[0249]

Figure 1

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Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Modes for Carrying Out the Invention

[0250] Hereinafter, the present invention will be further described through the description of specific embodiments, which does not limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concepts and scope of the present disclosure. Regarding the reagents and equipment used, those not described by the manufacturer are all commercially available conventional products.

[0251]

Table 12-1

Table 12-2

Table 13-1

Table 13-2

Table 13-3

[0252] Next, the present disclosure will be described by way of examples (which is not intended to limit the present disclosure). Referring to the following examples, the present disclosure will be described.

[0253] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present disclosure are substantially carried out according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Current Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will understand that the examples are illustrative of the present disclosure and are not intended to limit the scope of protection claimed by the present disclosure.

[0254] The abbreviations in the present disclosure have the following meanings. Abbreviations for which no meaning is provided have the meanings generally understood in the art.

Table 14

[0255] Example 1 Screening and Preparation of Anti-c-Met Antibody 1.1 Antigen information 1) Plasmid: The human cMet cDNA ORF Clone was purchased from Sino Biological Inc. (Catalog number HG10463-CH), and the nucleotide sequence corresponding to amino acids 25 - 932 of the extracellular segment of the human cMet protein was cloned into the pTT5-mFc and pTT5-hFc vectors (from Sichuan Sibowo Biotechnology Co., Ltd.), and finally expression plasmids were formed by fusing the human IgG Fc segment and the mouse IgG Fc segment respectively.

[0256] 2) Antigen expression: HEK293E cells (from Sichuan Sibowo Biotechnology Co., Ltd.) were transiently transfected with PEI max (Polysciences, 24765-1) and expressed for 7 days. Then, by purification with ProA filler (GE, Mabselect XL), the hcMet-ECD-mFc and hcMet-ECD-hFc antigen proteins were obtained.

[0257] 3) Commercially available antigen proteins: Human HGF R / c-MET Protein (Fc Tag) is from Bepsis (Catalog number: MET-H5256), Human HGF R / c-MET Protein (His Tag) is from Bepsis (Catalog number: MET-H5227), c-MET Protein, Cynomolgus, Rhesus, Recombinant (His Tag) is from Sino Biological (Catalog number: 90304-C08H), Rat-cMet-hFc, c-MET Protein, Rat, Recombinant (hFc Tag) is from Sino Biological (Catalog number: 80004-R02H), mouse cMet-ECD-His, c-MET Protein, Mouse, Recombinant (ECD, is Tag) is from Sino Biological (Catalog number: 50622-M08H), and HGF Protein, Human, Recombinant is from Sino Biological (Catalog number: 10463-HNAS).

[0258] 1.2 Immunity and antibody screening Four mice each were selected from the CD1 (Vital River), KM (Vital River), and Balb / c (Jicui Bio) mouse strains. All were female and around 6 weeks old and were grouped. Immunization was performed using cMET-His and hcMET-mFc (Sichuan Sibowo Biotechnology Co., Ltd.). After two booster immunizations, the serum titer was measured by ELISA. For mice with high ELISA protein titers, the tumor cell MKN-45 affinity titer and MKN45-HGF competitive FACS titer were detected by FACS. Finally, the spleens and lymph nodes of one mouse each from CD1, KM, and Balb / c were selected to prepare cell suspensions, and hybridoma fusion was performed with SP2 / 0 mouse myeloma cells. Initial screening was performed by ELISA with the human hMET-ECD-hFc antigen protein. After initial screening, positive hybridomas were selected and cloned. The hybridoma cells were cultured in serum-free medium, and the collected monoclonal supernatants were purified by a ProA filler to obtain mouse-derived antibodies. The affinity between the mouse-derived antibodies and human MET-ECD-hFc and cynomolgus monkey MET-ECD-hFc proteins was detected by ELISA, and the affinity between the mouse-derived antibodies and MKN45 tumor cells was detected by FACS to block the MKN45-HGF ligand competition. After multiple screenings, a total of 18 positive clones were obtained, and 18 mouse-derived antibodies were evaluated. The evaluation results of the mouse-derived antibodies of the four clones with high affinity are as shown in Table 1.

Table 15

[0259] The sequences were captured for the four high-affinity monoclonal antibodies 55A10G6, 45A5G10, 51D5B2, and 44H10E8. The method for capturing the sequences is as follows: approximately 1×10 5Collect the candidate hybridoma cells, extract RNA with Trizol, perform reverse transcription via PolyA using the PrimeScript RT reagent kit to obtain cDNA, design forward primers upstream of the heavy chain and light chain respectively, and design reverse primers downstream of the heavy chain CH1 region and light chain CL region. Amplify the product by PCR, recover the fragment via the agarose gel recovery kit, send the sample to Beijing Tsingke Biotechnology Co., Ltd for sequencing, and refer to the sequence information table for the specific sequences of the mouse-derived antibody variable region and CDR.

[0260] 1.3 Humanization of the antibody Adopt the CDR grafting method. First, use the conventional BLAST method to find the human germline sequence with the highest homology to the original mouse-derived sequence as a template, transplant the CDR of the mouse-derived antibody onto the human template to construct a chimera, analyze the FR amino acids that can retain the original three-dimensional structure in the mouse-derived antibody according to the structure, perform back mutations on the corresponding amino acids in the chimera to mouse-derived amino acids to maintain the original affinity, perform computational and immunogenicity analyses on the constructed humanized antibody, find highly immunogenic fragments, and substitute them with low immunogenic fragments. Regarding the substitution of highly immunogenic sites within the CDR, for example, in humanization, substitute 45A5G10 Kabat HCDR2, QIRLKSLNYATHYA E from SVKG (SEQ ID NO:17) to QIRLKSLNYATHYA Q substitute 55A10G6 Kabat HCDR2, WIFPGSGNTKY IE KF K from G(SEQ ID NO:30) to WIFPGSGNTKY SQ KF QIt was replaced with G (SEQ ID NO:31). Humanization was performed on mouse-derived antibodies 55A10G6 (abbreviated as 55A10G6) and 45A5G10 (abbreviated as 45A5G10) to obtain humanized variable regions 55A10G6-HZ VH, 55A10G6-HZ VL, 45A5G10-HZ VH, and 45A5G10-HZ VL. The specific sequences of the variable regions and CDRs of each humanized antibody are referred to in the sequence information table. Here, the CDRs are provided according to the IMGT, chothia, and Kabat definition schemes.

[0261] 1.4 Expression of anti-c-Met humanized antibody The heavy-chain variable region 55A10G6-hz vh of the humanized antibody was ligated to the heavy-chain IgG1 constant region (SEQ ID NO:51), and the light-chain variable region 55A10G6-hz vl of the humanized antibody was ligated to the Kappa constant region (SEQ ID NO:52). The obtained ligated sequence was submitted to a general-purpose organism for gene synthesis. After codon optimization, it was constructed into a PTT5 vector. After plasmid synthesis, HEK293E cells (from Sichuan Sibowo Biotechnology Co., Ltd.) were transfected with PEImax, expressed for about 7 days, and centrifuged to collect the supernatant. The supernatant was purified using a ProA filler. All of the purified antibody was ultrafiltered with PBS buffer to measure the concentration and stored at -20°C. Antibody 55A10G6-hz was obtained.

[0262] The heavy-chain variable region 45A5G10-hz vh of the humanized antibody was ligated to the heavy-chain IgG1 constant region (SEQ ID NO:51), and the light-chain variable region 45A5G10-hz vl of each humanized antibody was ligated to the Kappa constant region (SEQ ID NO:52). The obtained ligated sequence was submitted to a general-purpose organism for gene synthesis. After codon optimization, it was constructed into a PTT5 vector. After plasmid synthesis, HEK293E cells were transfected with PEImax, expressed for about 7 days, and centrifuged to collect the supernatant. The supernatant was purified using a ProA filler. All of the purified antibody was ultrafiltered with PBS buffer to measure the concentration and stored at -20°C. Antibody 45A5G10-hz was obtained.

[0263] The amino acid sequences of the heavy and light chain variable regions of the control antibody (ABT700, sequence from KEGG ID: D11307) were submitted to a general organism for gene synthesis. After codon optimization, they were constructed into the PTT5 vector. After plasmid synthesis, HEK293E cells were transfected with PEImax, expressed for about 7 days, and centrifuged to collect the supernatant. The supernatant was purified using a ProA filler. All of the purified antibody was ultrafiltered into PBS buffer, the concentration was measured, and it was stored at -20°C.

[0264] Example 2 Evaluation of anti-c-Met antibody 2.1 ELISA affinity evaluation of anti-c-Met antibody protein The antigen protein cMet-ECD-his was diluted with carbonate buffer (CBS) at 1 μg / ml, and the antigen was coated. Then, it was blocked with 2% BSA (in phosphate-buffered PBS) at 37°C for 2 hours. Serial diluted humanized antibodies to be tested (starting from 2 μg / mL, 3-fold dilution, 11 concentration points) were added, incubated at 37°C for 2 hours, HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164) was added, incubated at 37°C for 1 hour, TMB substrate was added to develop color, and after termination with 2M HCl, the absorbance value at 450 nM was read by a machine. The antigen protein anti-His-Rabbit Fc (from Chengdu NB Biolab Co., Ltd.) was diluted with CBS at 1 μg / ml, and the antigen was coated. Then, it was blocked with 2% BSA (in PBS) at 37°C for 2 hours. 0.5 μg / ml cyno.cMet-ECD-His was added and incubated at 37°C for 2 hours. Serial diluted humanized antibodies to be tested (starting from 2 μg / mL, 3-fold dilution, 11 concentration points) were added for 2 hours and incubated at 37°C for 2 hours. HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164) was added, incubated at 37°C for 1 hour, TMB substrate was added to develop color, and after termination with 2M HCl, the absorbance value at 450 nM was read by a computer.

[0265] The experimental results are shown in Table 2. The two humanized antibodies both had high affinity for human and cynomolgus monkey Met proteins, and showed significantly stronger affinity for cynomolgus monkey Met than ABT700.

Table 16

[0266] 2.3 Flow affinity evaluation of anti-c-Met antibodies MKN45 cells (Nanjing Kebai, CBP60488) were collected by trypsin digestion and centrifugation, washed three times with pre-cooled PBS, resuspended in 1% BSA (in PBS), and 2^10 5 cells (50 ul) were spread in a 96-well V-bottom plate. Starting from 20 ug / ml and with a 4-fold dilution gradient and eight dilution points, the anti-cMet humanized antibody to be tested was serially diluted with 1% BSA. 50 ul of the diluted antibody was uniformly mixed with the cells in the V-bottom plate, incubated at 4°C for 1 hour, washed three times with pre-cooled PBS, and then 100 ul of 1% BSA (containing 1 ul of anti-human APC fluorescent secondary antibody, BioLegend, catalog number 410712) was added to each well, incubated at 4°C for 0.5 hour, washed three times with pre-cooled PBS, and then the cells were resuspended and detected by flow cytometry (Beckman, cytoflex).

[0267] The experimental results are shown in Table 3. The two humanized antibodies and MKN45 cells both had high affinity, and the affinity of the two antibodies for MKN45 was higher than that of the control antibody ABT700.

Table 17

[0268] 2.4 Kinetic affinity evaluation of anti-c-Met antibodies The dynamic affinity of the humanized antibody was measured by ForteBio. The experimental procedure was as follows: 1. Preparation of the sensor: Take out the ProA sensor, pre-wet the sensor with PBST diluent (pH 7.4) for 10 minutes. 2. Dilution of the sample: Dilute the antibody to be immobilized to 5 μg / ml each, and start with the antigen h.cMet-ECD-His (BPS, catalog number: MET-H5227) at 500 nM, perform serial two-fold dilutions at five concentration points, and set a 0 concentration point. 3. Program setting: Insert the sensor plate and the sample plate, start the program, and regenerate the sensor with 20 mM glycine solution (pH 1.7). 4. Analyze the data using Octet analysis software. The experimental results are shown in Table 4. Antibody 45A5G10-Hz, antibody 55A10G6-Hz, and human cMet protein all have high dynamic affinity.

Table 18

[0269] 2.5 Identification of the quality of anti-c-Met antibodies 1) Identify the purity of the humanized antibody by SEC, and the detection method is as follows: Equipment: Waters Alliance e2695 HPLC; Chromatography column: Thermo MabPac SEC-1, 5 μm, 7.8 * 300 mm; Mobile phase: 61 mmol / L Na 2 HPO 4 、39 mmol / L NaH 2 PO 4 、200 mmol / L NaCl, 5% IPA; Instrument parameters: Sample chamber temperature: 8 °C; Column temperature: 30 °C; Flow rate: 0.5 ml / min; Injection volume: 20 μg; Detection wavelength: 280 nm; Isocratic operation: 30 min.

[0270] 2) Detect the hydrophilicity and hydrophobicity of the humanized antibody by HIC. The detection method is as follows: Use Tosoh's hydrophobic chromatography column (TOSOH Tskgel Buty-NPR(2.5), 4.6*100), and perform the detection of hydrophilicity and hydrophobicity with an Agilent HPLC device. Mobile phase A is 1.5M (NH 4 ) 2 SO 4 and mobile phase B is 25mM Na 2 HPO 4 (pH = 7.0) + 25% IPA. The device parameters were set as follows: sample chamber temperature: 8 °C, column temperature: 30 °C, flow rate: 0.5 mL / min, detection wavelength: 280 nm. The test humanized antibody sample was diluted to a final concentration of 1 mg / mL with mobile phase A, and 20 μL was injected for gradient elution.

[0271] 3) Measure the Tm value of the humanized antibody using DSF, which reflects the thermal stability of the antibody. The experimental procedure is as follows: Dilute the test humanized antibody sample to 1 mg / mL with PBS, dilute the dye SYPRO Orange dye (Thermo #56651) 40X with ddH 2 O, and the reaction system: sample 12.5 μL + 40X dye 2.5 μL + ddH 2 O 5 μL. Seal the membrane and perform instantaneous centrifugation. By Q-PCR detection, the Q-PCR parameter settings are: Target (ROX), program (25 °C, 3 min, 1% rate, 95 °C, 95 °C, 2 min).

[0272] The detection results of SEC, HIC, and Tm value are shown in Table 5. The results of SEC show that the purity of both humanized antibodies is high, greater than 90%. The results of HIC show that the binding of both humanized antibodies to the hydrophobic chromatography column is weak, the retention time is short, and the hydrophilicity of both is good. The Tm value shows that both humanized antibodies have good thermal stability.

Table 19

[0273] 2.6 Cross-detection of anti-c-Met antibodies with rat and mouse cMet Dilute the antigen protein Rat-cMet-hFc to 0.5 μg / ml with CBS, coat the antigen, block it with 2% BSA (in PBS) at 37°C for 2 hours, add the serially diluted test humanized antibody (biotin-labeled) (starting from 2 μg / mL, 3-fold dilution, 11 concentration points), incubate at 37°C for 2 hours, add the HRP-labeled anti-biotin secondary antibody (proteintech, sa00001-0), incubate at 37°C for 1 hour, add the TMB substrate for color development, and after terminating with 2M HCl, read the absorbance value at 450 nM with a machine.

[0274] Dilute the protein anti-His-hFc (from Chengdu NB Biolab Co., Ltd.) to 0.5 μg / ml with CBS, then coat it, block it with 2% BSA (in PBS) at 37°C for 2 hours, add 1 μg / ml of mouse cMet-ECD-His, incubate at 37°C for 2 hours, add the serially diluted test humanized antibody (starting from 2 μg / mL, 3-fold dilution, 11 concentration points) for 2 hours, incubate at 37°C for 2 hours, add the HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164), incubate at 37°C for 1 hour, add the TMB substrate for color development, and after terminating with 2M HCl, read the absorbance value at 450 nM with a machine.

[0275] The experimental results are shown in Figure 1 and Figure 2. The two humanized antibodies, 45A5G10-Hz and 55A10G6-Hz, do not cross-react with either rat or mouse cMet (neither binds to rat or mouse cMet).

[0276] 2.7 Evaluation of the endocytosis activity of anti-c-Met antibodies Collect MKN45 cells by trypsin digestion and centrifugation, incubate the test humanized antibody at a concentration of 10 μg / mL at 4°C for 1 hour, wash 3 times with PBS, resuspend the cells with pre-warmed DMEM + 10% FBS at 37°C, divide into three parts and incubate at 37°C for 0, 2, and 4 hours respectively, wash 3 times with pre-cooled PBS, then add 1.2 μL of anti-human APC fluorescent secondary antibody, incubate at 4°C for 0.5 hour, wash 3 times with PBS, and then resuspend the cells and place them on the machine. Calculate the endocytosis rate according to the following formula: Endocytosis rate (%) = [1 - (average fluorescence value of the test sample at that time - average fluorescence value of the negative control sample at that time) / (average fluorescence value of the test sample at 0 hour - average fluorescence value of the negative control sample at 0 hour)] * 100.

[0277] The experimental results are shown in Table 6. The endocytosis activities of antibodies 45A5G10-Hz and 55A10G6-Hz at the same time were both higher than that of ABT700, and the endocytosis rate could reach more than 60% at 4 hours.

Table 20

[0278] 2.8 Evaluation of epitope competition of anti-c-Met antibody Coat the CBS with anti-His-Rabbit Fc protein at 0.5 μg / ml, block it with 2% BSA (in PBS) at 37 °C for 2 hours, add 1 μg / ml of h.cMet-ECD-His and incubate at 37 °C for 2 hours. After 2 hours, add the test humanized antibody (starting from 10 μg / mL, 3-fold dilution, 11 concentration points) diluted in gradient so that the final concentration starts from 5 μg / ml, incubate at room temperature for 0.5 hour. After the incubation is completed, add 200 ng / ml of ABT700-Biotin so that the final concentration becomes 100 ng / ml, incubate at room temperature for 2 hours. After 2 hours, add the HRP-labeled anti-biotin secondary antibody (proteintech, sa00001-0), incubate at 37 °C for 1 hour, add the TMB substrate to develop color, and after terminating with 2M HCl, read the absorbance value at 450 nM with a machine.

[0279] The experimental results are shown in Figure 3. There is no epitope competition between the two humanized antibodies 45A5G10-Hz and 55A10G6-Hz and ABT700.

[0280] 2.9 Evaluation of the competition between anti-c-Met antibody and HGF ligand cMET is a tyrosine kinase receptor expressed on the cell membrane, binds to the ligand HGF via the Sema domain, thereby triggering a downstream phosphorylation cascade reaction, and ultimately promoting cell proliferation. Thus, the competitive effect of the humanized antibody on ligand binding and the effect of inhibiting HGF-induced ERK phosphorylation were detected.

[0281] Collect MKN45 cells by trypsin digestion and centrifugation, wash them 3 times with pre-cooled PBS, resuspend the cells in 1% BSA (in PBS), and 2^10 per well 5Spread individual cells (50 μl) on a 96-well V-bottom plate. Gradient dilute the anti-cMet humanized antibody with 1% BSA, starting from 15 μg / ml, with the final concentration starting from 5 μg / ml, and perform a 3-fold dilution gradient, with 8 dilution points. Mix 50 μl of the diluted antibody homogeneously with the cells in the V-bottom plate. Dilute HGF-His-biotin (from Chengdu NB Biolab Co., Ltd.) to 150 ng / ml with 1% BSA, take 50 μl / well and add it to the well plate, making the final concentration of the ligand 50 ng / ml. After thorough mixing, incubate at 4°C for 1 hour. After washing 3 times with pre-cooled PBS, add 100 μl of 1% BSA (containing 1 μl of anti-biotin PE fluorescent secondary antibody, BioLegend, catalog number 405204) to each well, incubate at 4°C for 0.5 hour, wash 3 times with pre-cooled PBS, resuspend the cells and detect by flow cytometry (Beckman, cytoflex).

[0282] The experimental results are shown in Table 7. Both of the two humanized antibodies have competitive activity with the HGF ligand on MKN45 cells, and the competitive activity is higher than that of ABT700.

Table 21

[0283] 2.10 Inhibition of HGF-induced ERK phosphorylation by anti-c-Met antibody MDA-MB-468 cells (ATCC, catalog number HTB-132) were counted by trypsin digestion, and the cells were resuspended in L15 medium at 2×10^5 / ml. Then the cells were spread on a 24-well plate, and the cell suspension was adjusted to 1 ml per well, with a total of 4 wells. After that, the cells were returned to the cell culture medium at 37°C and left overnight for starvation culture. After 16 hours, the cells in the 6 wells were grouped and set as untreated, HGF (Sino Biological, catalog number 10463-HNAS), HGF + isotype control (hIgG1, prepared by Suzhou Eli Biopharmaceuticals Co., Ltd.), HGF + 45A5G10-Hz, and HGF + 55A10G6-Hz. Then, the corresponding sterile samples were added to the corresponding cell wells, and the test antibody was added alone 15 minutes in advance (final concentration 15 μg / ml). After 15 minutes, HGF was added to a final concentration of 100 ng / ml, and the cells were returned to the cell culture solution at 37°C and statically cultured for 15 minutes. After 15 minutes, the cells were washed 3 times with PBS, and SDS lysis solution was directly added to each well to lyse the cells and recover the proteins. Conventional SDS-PAGE and Western blot detection were performed on the obtained proteins. Here, the detection antibody for phosphorylated ERK was obtained from CST (catalog number 5726S), and the GAPDH detection antibody was obtained from Sino Biological (catalog number 100242-MM05). The dilution ratios of the two primary antibodies were both 1:2000, and they were incubated overnight at 4°C.

[0284] The results are shown in Figure 4. Treatment with HGF alone can significantly induce ERK phosphorylation in MDA-MB-468 cells. However, by pre-adding antibodies 45A5G10-Hz and 55A10G6-Hz, HGF-induced ERK phosphorylation can be completely blocked.

[0285] 2.11 Induction of cMet protein degradation by humanized antibodies The MKN-45 cells were counted by trypsin digestion, and the cells were resuspended in RPMI 1640 + 10% FBS (fetal bovine serum) + Ps (penicillin-streptomycin) to 2*10^5 / ml. Then, the cells were spread on a 24-well plate, with 1 ml of cell suspension per well, for a total of 4 wells. The cells were returned to the cell culture medium at 37°C and allowed to stand overnight. After 16 hours, the cells in the 4 wells were grouped and set up as untreated, isotype control (hIgG1), 45A5G10-Hz, and 55A10G6-Hz, respectively. The corresponding sterile samples were added to the corresponding cell wells, and the final concentration of the antibody was 20 ug / ml. The cells were returned to the cell culture medium at 37°C and incubated statically for 60 hours. After 60 hours, the cells were washed 3 times with PBS, and SDS lysis solution was directly added to each well to lyse the cells and recover the protein. Conventional SDS-PAGE and Western blot detection were performed on the obtained protein. The detection antibody for cMet was obtained from ProteinTech (catalog number 25869-1-AP), and the detection antibody for GAPDH was obtained from Sino Biological (catalog number 100242-MM05). The dilution ratios of the two primary antibodies were both 1:2000, and they were incubated overnight at 4°C.

[0286] The results in Figure 5 show that the anti-cMet antibodies 45A5G10-Hz and 55A10G6-Hz can significantly induce the degradation of cMet compared to the treatment with the isotype control antibody IgG1.

[0287] Example 3 Preparation of Anti-c-Met ADC Example 3.1 Preparation of Anti-c-MET-B81 ADC

Chemical formula

[0288] 3.1.1 Preparation of 45A5G10-HZ-B81 (DAR8) Sample A total of 20 mg of the anti-c-Met antibody 45A5G10-HZ was added to a sodium edetate solution with a final concentration of 1 mM, and mixed well. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution. A 20 mmol / L TCEP solution with a molar equivalent of 6.5 times the antibody was added, and mixed well. The mixture was left at 37 °C for 90 minutes. To the above solution system, 10 mmol / L B81 (prepared by referring to DL-037 in Example 2.37 of WO2022170971) dissolved in dimethyl sulfoxide with a molar equivalent of 15 times the antibody was added, and mixed well. The mixture was left at 37 °C for 3 hours to obtain a post-binding sample. After the reaction was completed, the sample was replaced with 20 mM histidine with a pH of 6.0 using a 30 KDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC 45A5G10-HZ-B81 (DAR8).

[0289] Measurement of the DAR value of the post-binding sample by RP-LC / MS

Table 22

Table 23

[0290] The results are shown as follows. The light chain of the 45A5G10-HZ-B81 (DAR8) sample bound to 0 to 1 toxin molecule (LC, DAR1 ratios were 0% and 100.0% respectively), and the heavy chain bound to 0 to 3 toxin molecules (the ratios of mAb, DAR1, DAR2, and DAR3 were 0%, 0%, 0%, and 100.0% respectively). Thereby, the binding ratio (DAR value) of the 45A5G10-HZ-B81 (DAR8) sample was calculated to be 8.0. It can be inferred that q is 8.

[0291] The above mAb represents an unbound monoclonal antibody, LC represents the light chain of the antibody, HC represents the heavy chain of the antibody, DAR1 represents a complex containing a light chain or heavy chain bound to one toxin molecule, DAR2 represents a complex containing a light chain or heavy chain bound to two toxin molecules, DAR3 represents a complex containing a light chain or heavy chain bound to three toxin molecules, where the theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform. The same applies to the following mAb, LC, HC, DAR1, DAR2, and DAR3.

[0292] 3.1.2 Preparation of 45A5G10-HZ-B81 (DAR4) sample A total of 20 mg of anti-c-Met antibody 45A5G10-HZ was added to a sodium edetate solution with a final concentration of 1 mM, mixed well, the pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution, a 20 mmol / L TCEP solution with a molar equivalent 2.7 times that of the antibody was added, mixed well, left standing at 5 °C for 5 h, 10 mmol / L B81 (prepared by referring to DL-037 in Example 2.37 of WO2022170971) dissolved in dimethyl sulfoxide with a molar equivalent 6.5 times that of the antibody was added to the above solution system, mixed well, left standing at room temperature for 1 h to obtain a post-binding sample. After the reaction was completed, the sample was replaced with 20 mM histidine buffer with a pH of 6.0 using a 30 KDa ultrafiltration tube to remove low-molecular-weight substances, and finally the sample was concentrated to obtain a solution containing anti-c-Met antibody ADC 45A5G10-HZ-B81 (DAR4).

[0293] Measurement of DAR value of post-binding sample by Native-LC / MS [Table 24] [Table 25]

[0294] The results are shown as follows. The whole antibody of the 45A5G10-HZ-B81 (DAR4) sample binds to 0 to 8 toxin molecules (the ratios of mAb, q = 2, q = 4, q = 6, q = 8 are 0%, 22%, 56%, 23%, 0% respectively), and thereby the binding ratio (DAR value) of the 45A5G10-HZ-B81 (DAR4) sample is calculated to be 4.0.

[0295] 3.1.3 Preparation of 55A10G6-HZ-B81 (DAR8) A total of 20 mg of the anti-c-Met antibody 55A10G6-HZ was added to a sodium edetate solution with a final concentration of 1 mM, mixed well, the pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution, a 20 mmol / L TCEP solution with a 6.5-fold molar equivalent of the antibody was added, mixed well, left at 37 °C for 90 minutes, 10 mmol / L B81 dissolved in dimethyl sulfoxide with a 15-fold molar equivalent of the antibody was added to the above solution system, mixed well, left at 37 °C for 3 hours to obtain a sample after binding. After the reaction was completed, the sample was replaced with a 20 mM histidine buffer with a pH of 6.0 using a 30 KDa ultrafiltration tube to remove low molecular weight substances, and finally the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC 55A10G6-HZ-B81 (DAR8).

[0296] Referring to the LC / MS in the above 3.1.1, the DAR value of the sample after binding was measured. The light chain of the 55A10G6-HZ-B81 sample binds to 0 to 1 toxin molecule (the ratios of LC, DAR1 are 0%, 100.0% respectively), and the heavy chain binds to 0 to 3 toxin molecules (the ratios of mAb, DAR1, DAR2, DAR3 are 0%, 0%, 0%, 100.0% respectively), and thereby the binding ratio (DAR value) of the 55A10G6-HZ-B81 sample was calculated to be 8.0. It can be inferred that q is 8.

[0297] Example 3.2 Preparation of anti-c-MET-vc-MMAE

Chemical formula

[0298] 3.2.1 Preparation of ABT700-vc-MMAE (DAR9.7) Sample A total of 20 mg of anti-c-Met antibody ABT700 was added to a sodium edetate solution with a final concentration of 1 mM, mixed well, the pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution, a 20 mmol / L TCEP solution of 6.5 molar equivalents of the antibody was added, mixed well, left at 37 °C for 90 minutes, 10 mmol / L vc-MMAE (CAS No.: 646502-53-6, purchased from MedChemExpress) dissolved in dimethyl sulfoxide of 15 molar equivalents of the antibody was added to the above solution system, mixed well, left at 37 °C for 3 hours to obtain a post-binding sample. After the reaction was completed, the sample was replaced with a 20 nM histidine buffer with a pH of 6.0 using a 30 KDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC ABT700-vc-MMAE (DAR9.7).

[0299] Referring to the LC / MS in the above 3.1.1, the DAR value of the post-binding sample was measured. The light chain of the ABT700-Vc-MMAE (DAR9.7) sample bound to 1 to 3 toxin molecules (the ratios of DAR1, DAR2, and DAR3 were 64.3%, 29.8%, and 5.9% respectively), and the heavy chain bound to 3 to 4 toxin molecules (the ratios of DAR3 and DAR4 were 56.4% and 43.6% respectively). Thereby, the binding ratio (DAR value) of the ABT700-Vc-MMAE (DAR9.7) sample was calculated to be 9.7.

[0300] 3.2.2 Preparation of ABT700-vc-MMAE (DAR4) Sample A total of 20 mg of the anti-c-Met antibody ABT700 was added to a sodium edetate solution with a final concentration of 1 mM, and mixed well. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution. A 20 mmol / L TCEP solution at 2.8 molar equivalents of the antibody was added and mixed well. The mixture was left at room temperature for 90 minutes. Then, a 10 mmol / L vc-MMAE (CAS No.: 646502-53-6, purchased from MedChemExpress) dissolved in dimethyl sulfoxide at 4.2 molar equivalents of the antibody was added to the above solution system and mixed well. The mixture was left at room temperature for 3 hours to obtain a post-binding sample. After the reaction was completed, the sample was replaced with a 20 mM histidine buffer with a pH of 6.0 using a 30 KDa ultrafiltration tube to remove low-molecular-weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC ABT700-Vc-MMAE (DAR4).

[0301] Referring to the LC / MS in 3.1.1 above, the DAR value of the post-binding sample was measured. The light chain of the ABT700-VC-MMAE (DAR4) sample bound to 0 to 1 toxin molecule (the ratios of LC and DAR1 were 52.2% and 47.8% respectively), and the heavy chain bound to 0 to 3 toxin molecules (the ratios of mAb, DAR1, DAR2 and DAR3 were 17.5%, 47.2%, 22.9% and 12.5% respectively). Thereby, the binding ratio (DAR value) of the ABT700-VC-MMAE (DAR4) sample was calculated to be 4.0.

[0302] Example 4 In Vitro Evaluation of Anti-Human c-Met Antibody and Its ADC 4.1 Detection of Affinity of Anti-Human cMet ADC Protein Coat CBS with 0.5 μg / ml of the protein anti-His-RabbitFc, block it with 2% BSA (in PBS) at 37°C for 2 hours, add 1 μg / ml of h.cMet-ECD-His and incubate at 37°C for 2 hours. After 2 hours, add the serially diluted test antibody or ADC (starting from 2 μg / mL, 3-fold dilution, 11 concentration points), incubate at 37°C for 2 hours. After 2 hours, add the HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164), incubate at 37°C for 1 hour, add the TMB substrate to develop color, and after terminating with 2M HCl, read the absorbance value at 450 nM with a machine.

[0303] The experimental results are shown in Table 8. There are no obvious changes in the antigen-binding affinity of 45A5G10-HZ, 45A5G10-HZ-B81 (DAR8), 55A10G6-Hz, and 55A10G6-HZ-B81 (DAR8), but the antigen-binding affinity clearly decreases after ABT700 binds to the ADC.

Table 26

[0304] 4.2 Detection of the affinity of anti-human cMet ADC cells Test 1 Collect NCI-H358 (human non-small cell lung cancer cells, purchased from ATCC, catalog number CRL-5807), LS1034 cells (human colon adenocarcinoma cells, Nanjing Kebai, catalog number CBP60013), NCI-H69 (human small cell lung cancer cells, Wuhan Punuosai, catalog number CL-0677), and NCI-H716 (human colorectal adenocarcinoma cells, purchased from ATCC, catalog number CCL-251) by trypsin digestion and centrifugation. Collect the cells directly by centrifugation, wash them 3 times with pre-cooled PBS, resuspend the cells in 1% BSA (in PBS), and add 2*10 per well 5Spread the cells (50 μl) in a 96-well V-bottom plate. Dilute the ADC of the anti-cMet humanized antibody starting from 15 μg / ml, with a 4-fold dilution gradient and 8 dilution points, using 1% BSA for the dilution gradient. Mix 50 μl of the diluted ADC with the cells in the V-bottom plate, incubate at 4°C for 1 hour, wash 3 times with pre-cooled PBS, then add 100 μl of 1% BSA (containing 1 μl of anti-human APC fluorescent secondary antibody, BioLegend, catalog number 410712) to each well, incubate at 4°C for 0.5 hour, wash 3 times with pre-cooled PBS, and then resuspend the cells for detection by flow cytometry (Beckman, cytoflex).

[0305] The experimental results are shown in Table 9. After the two humanized antibodies bound to B81, the ADCs showed high affinity for NCI-H358, LS1034, NCI-H69, and NCI-H716 cells, and the affinity of the ADCs of the two antibodies for the three tumor cells was higher than that of ABT700-VcMMAE (DAR9.7). The affinity of the ADCs of the two antibodies for NCI-H716 cells was higher than that of ABT700-VcMMAE (DAR4).

Table 27

[0306] Test 2 Collect MKN45 (from JCBR JCRB0254) cells by trypsin digestion and centrifugation, wash 3 times with pre-cooled PBS, and dilute the cells to 1×10 6 cells / mL, with 1×10 5Spread the cells (100 μL) in a 96-well V-bottom plate. Dilute 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ starting from 25 nM, with a 4-fold dilution gradient and 8 dilution points, using FACS buffer for the dilution gradient. Mix with the cells in the V-bottom plate at 100 μL / well, incubate at 4 °C for 0.5 h, wash 3 times with pre-cooled PBS, add 100 μL of fluorescent secondary antibody PE anti-human IgG diluted with ice-cold FACS buffer to each well, incubate at 4 °C for 0.5 h, wash 3 times with pre-cooled PBS, then resuspend the cells and detect by flow cytometry.

[0307] The results are shown below. 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ have high binding activity to MKN45. 45A5G10-HZ and 45A5G10-HZ-B81 (DAR8) have equivalent affinity for MKN45, and the EC 50 are 0.215 nM and 0.215 nM, respectively.

[0308] 4.3 Detection of in vitro killing activity of ADC Test 1 Digest tumor cells in good growth state and centrifuge to collect SW480 (purchased from ATCC, catalog number CCL-228), NCI-H358 (purchased from ATCC, catalog number CRL-5807), LS1034 cells (Nanjing Kebai, catalog number CBP60013), and NCI-H716 (purchased from ATCC, catalog number CCL-251). Centrifuge the cells directly to collect them. Resuspend all the above cells in RPMI1640 + 5% FBS + Ps, count them, and spread 5000 cells (LS1034 and NCI-H716) or 3000 cells (SW480 and NCI-H358) per well with a volume of 100 uL. Dilute the ADC to be tested starting from 300 μg / mL (2 μM) in the resuspension medium corresponding to the above three types of cells, perform 3-fold dilution at 11 concentration points, take 100 μL / well and add it to the plate so that the final ADC concentration starts from 150 μg / mL. Incubate at 37°C for 5 days (LS1034, NCI-H358) or 7 days (NCI-H716, SW480). After the incubation is completed, add 20 μL / well of CCK8, react for 1 - 3 hours, read at 450 nm with a microplate reader, and import it into Graphpad Prism for curve fitting.

[0309] The experimental results are shown in Table 10. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) can effectively kill four tumor cells, namely LS1034, NCI-H358, NCI-H716, and SW480. In LS1034 cells, the cytotoxic activity of 45A5G10-Hz-B81 (DAR8) is equivalent to that of ABT700-VcMMAE (DAR9.7), while 55A10G6-Hz-B81 (DAR8) is slightly weaker than ABT700-VcMMAE (DAR9.7). In NCI-H358 cells, the killing activity of 45A5G10-Hz-B81 (DAR8) is stronger than that of 55A10G6-Hz-B81 (DAR8). In NCI-H716 cells, the killing activities of both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) are stronger than that of ABT700-VcMMAE (DAR4). In SW480 cells, the cytotoxic activities of 45A5G10-Hz-B81 (DAR8) and ABT700-VcMMAE (DAR4) are stronger, while 55A10G6-Hz-B81 (DAR8) is equivalent to ABT700-VcMMAE (DAR4).

Table 28

[0310] Test 2 NCI-H358 cells (from ECACC-95111733), NCI-H441 cells (from ATCC-HTB-174), and MKN45 cells (from JCBR JCRB0254) were collected by trypsin digestion and centrifugation, resuspended in RPMI 1640 medium containing 10% FBS, counted, and 1500 (NCI-H441 / NCI-H358 / MKN45) cells were seeded into each well with a volume of 135 μL. Test article 45A5G10-HZ-B81 (DAR8) was diluted in the resuspension medium corresponding to the above three types of cells starting from 20000 nM, serially diluted 4-fold at 9 concentration points, and further diluted starting from 500 nM with the medium, then added to the plate at 15 μL / well to make the final concentration of the ADC 50 nM, and incubated at 37 °C for 6 days. After the incubation was completed, CellTiter-Glo working solution was added at 75 μL / well, shaken on an orbital shaker for 2 minutes to induce cell lysis, left at room temperature for 10 minutes to stabilize the luminescence signal, and the luminescence signal was detected with a microplate reader.

[0311] The experimental results are shown in Table 11, and the results indicate that 45A5G10-HZ-B81 (DAR8) has a strong anti-proliferative effect on NCI-H441 cells, with the corresponding IC50 being 0.031 nM. 45A5G10-HZ-B81 (DAR8) has an anti-proliferative effect on NCI-H358 cells, with the corresponding IC50 being 0.161 nM. 45A5G10-HZ-B81 (DAR8) has a strong anti-proliferative effect on MKN45 cells, with the corresponding IC50 being 0.057 nM.

Table 29

[0312] 4.4 Detection by Forte bio of the affinity of anti-c-Met antibodies and their ADCs for human Fc receptors and complement C1q ·4.4.1: Detection of the affinity of FcγRI with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ The Fc receptor FcγRI (also known as CD64) binds to the Fc terminus of IgG antibodies and can be involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to Fc receptors affects the safety and efficacy of such antibodies.

[0313] In this experiment, the Fortebio Octet molecular interaction apparatus was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRI, and to evaluate their potential ADCC and ADCP activities.

[0314] The experimental method for detecting the affinity constant between the corresponding antibody and FcγRI using the Fortebio Octet molecular interaction device is briefly described below. The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A solution of FcγRI (purchased from ACRO Biosystems, catalog number FCA-H52H1) at a concentration of 5 μg / mL was added to the HIS1K sensor to immobilize FcγRI on the sensor surface. Both the binding and dissociation parameters between the antibody and FcγRI were measured in the buffer, and the antibody concentrations were 200, 100, 50, 25, 12.5, and 6.25 nM. After equilibrating the sensor with the immobilized antigen in the buffer for 60 seconds, the binding between FcγRI immobilized on the sensor and each antibody was measured for 60 seconds, and the dissociation of FcγRI and the antibody was measured for 120 seconds. The data was analyzed by DataAnalysys11 to obtain the affinity constant between each antibody and FcγRI.

[0315] The measurement results of the affinity constants between FcγRI and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 12 below.

Table 30

[0316] ·4.4.2 Detection of the affinity between FcγRIIIa_V176 and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ The Fc receptor FcγRIIIa_V176 (also known as CD16a_V176) binds to the Fc terminus of IgG antibodies and can be involved in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0317] In this experiment, the Fortebio Octet molecular interaction apparatus was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIIa_V176, and their potential ADCC activities were evaluated.

[0318] The experimental method for detecting the affinity constant of the corresponding antibody with FcγRIIIa_V176 using the Fortebio Octet molecular interaction apparatus is briefly described below. The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A solution of FcγRIIIa_V176 (purchased from ACRO Biosystems, catalog number CD8-H52H4) at a concentration of 10 μg / mL was added to the HIS1K sensor to immobilize FcγRIIIa_V176 on the sensor surface. Both the binding and dissociation parameters of the antibody with FcγRIIIa_V176 were measured in the buffer, and the antibody concentrations were 2500, 1250, 625, 312.5, 156.25, and 78.125 nM. After equilibrating the sensor with the immobilized antigen in the buffer for 60 seconds, the binding of FcγRIIIa_V176 immobilized on the sensor with each antibody was measured for 60 seconds, and the dissociation of FcγRIIIa_V176 and the antibody was measured for 60 seconds. The data was analyzed by DataAnalysys11 to obtain the affinity constant of each antibody with FcγRIIIa_V176.

[0319] The measurement results of the affinity constants of FcγRIIIa_V176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 13 below.

Table 31

[0320] ·4.4.3 Detection of the affinity of FcγRIIIa_F176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ Fc receptor FcγRIIIa_F176 (also referred to as CD16a_F176) binds to the Fc terminus of IgG antibodies and can be involved in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0321] In the binding experiment of two samples with FcγRIIIa_F176 (purchased from ACRO Biosystems, catalog number CDA-H 522 0), except that the concentration range of the analyte in the binding step was optimized to (5000, 2500, 1250, 625, 312.5, 156.25 nM), the remaining steps were consistent with those of the CD16a (V176) binding test.

[0322] The measurement results of the affinity constants of FcγRIIIa_F176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 14 below.

Table 32

[0323] ·4.4.4 Detection of the affinity of FcγRIIa_H167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ Fc receptor FcγRIIa_H167 (also referred to as CD32a_H167) binds to the Fc terminus of IgG antibodies and can be involved in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0324] In this experiment, the Fortebio Octet molecular interaction apparatus was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIa_H167, and to evaluate their potential ADCC activities.

[0325] The experimental method for detecting the affinity constant between the corresponding antibody and FcγRIIa_H167 using the Fortebio Octet molecular interaction device is briefly described below. The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A solution of FcγRIIa_H167 (purchased from ACRO Biosystems, catalog number CD1 H5223) at a concentration of 10 μg / mL was added to the HIS1K sensor to immobilize FcγRIIa_H167 on the sensor surface. Both the binding and dissociation parameters between the antibody and FcγRIIa_H167 were measured in the buffer, and the antibody concentrations were 5000, 2500, 1250, 625, 312.5, and 156.25 nM. After equilibrating the sensor with the immobilized antigen in the buffer for 60 seconds, the binding between FcγRIIa_H167 immobilized on the sensor and each antibody was measured for 60 seconds, and the dissociation of FcγRIIa_H167 and the antibody was measured for 60 seconds. The data was analyzed using DataAnalysys11 to obtain the affinity constant between each antibody and FcγRIIa_H167.

[0326] The measurement results of the affinity constants between FcγRIIa_H167 and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 15 below.

Table 33

[0327] ·4.4.5 Detection of the affinity between FcγRIIa_R167 and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ The Fc receptor FcγRIIa_R167 (also referred to as CD32a_R167) can bind to the Fc terminus of IgG antibodies and be involved in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0328] In this experiment, the Fortebio Octet molecular interaction apparatus was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIa_R167, and their potential ADCC activities were evaluated.

[0329] The binding experiment of the two samples with FcγRIIa_R167 (purchased from ACRO Biosystems, catalog number CDA-H5221) was the same as the steps of the FcγRIIa_H167 binding experiment except for the binding step.

[0330] The measurement results of the affinity constants of FcγRIIa_R167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 16 below.

Table 34

[0331] ·4.4.6 Detection of the affinity of FcγRIIb / c with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ The Fc receptor FcγRIIb / c (also referred to as CD32b / c) can bind to the Fc terminus of IgG antibodies, negatively regulate the functions of immune cells, inhibit the activation and proliferation of immune cells, and inhibit the secretion of cytokines.

[0332] In this experiment, the Fortebio Octet molecular interaction apparatus was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIb / c, and evaluate the binding ability of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIb / c.

[0333] The experimental method for detecting the affinity constant between the corresponding antibody and FcγRIIb / c using the Fortebio Octet molecular interaction device is briefly described below. The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A solution of FcγRIIb / c (purchased from ACRO Biosystems, catalog number CDB-H5228) at a concentration of 10 μg / mL was added to the HIS1K sensor to immobilize FcγRIIb / c on the sensor surface. Both the binding and dissociation parameters between the antibody and FcγRIIb / c were measured in the buffer, and the antibody concentrations were 10000, 5000, 2500, 1250, 625, and 312.5 nM. After equilibrating the sensor with the immobilized antigen in the buffer for 60 seconds, the binding between FcγRIIb / c immobilized on the sensor and each antibody was measured for 60 seconds, and the dissociation of FcγRIIb / c from the antibody was measured for 60 seconds. The data was analyzed by DataAnalysys11 to obtain the affinity constant of each antibody and FcγRIIb / c.

[0334] The measurement results of the affinity constants between FcγRIIb / c and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 17 below.

Table 35

[0335] ·4.4.7 Detection of the affinity between FcRn and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ The Fc receptor FcRn binds to the Fc terminus of the IgG antibody, protects the antibody macromolecule from being destroyed, and releases the antibody macromolecule in the blood environment where the pH is 7.4.

[0336] In this experiment, the Fortebio Octet molecular interaction device was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcRn, and to evaluate the binding ability of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcRn.

[0337] The experimental method for detecting the affinity constant between the corresponding antibody and FcRn using the Fortebio Octet molecular interaction device is briefly described below. The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). An FcRn (purchased from ACRO) solution with a concentration of 2 μg / mL was added to the HIS1K sensor to immobilize FcRn on the sensor surface. Both the binding and dissociation parameters between the antibody and FcRn were measured in the buffer, and the antibody concentrations were 1000, 500, 250, 125, 62.5, and 31.25 nM. After equilibrating the sensor with the immobilized antigen in the buffer for 60 seconds, the binding between FcRn immobilized on the sensor and each antibody was measured for 60 seconds, and the dissociation of FcRn and the antibody was measured for 60 seconds. The data was analyzed by DataAnalysys11 to obtain the affinity constant of each antibody and FcRn.

[0338] The measurement results of the affinity constants of FcRn with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 18 below.

Table 36

[0339] ·4.4.8: Measurement of the affinity between C1q and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ Serum complement C1q can bind to the Fc terminus of IgG antibodies and mediate the CDC effect. The ability of a therapeutic monoclonal antibody to bind to C1q affects the safety and efficacy of the antibody.

[0340] In this experiment, the Fortebio Octet molecular interaction apparatus was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with C1q, and to evaluate the CDC activity of each antibody.

[0341] The experimental method for detecting the affinity constant between the corresponding antibody and C1q using the Fortebio Octet molecular interaction apparatus is briefly described below. The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). An antibody at 50 μg / mL was immobilized on the FAB2G sensor at a fixed height of approximately 3.0 nm, and the sensor was equilibrated in the buffer for 60 seconds. The antibody immobilized on the sensor was bound to the antigen C1q (purchased from Sigma, catalog number C1740-1MG). The 45A5G10-HZ antigen concentrations were 20, 10, 5, 2.5, 1.25, 0.625 nM, and the 45A5G10-HZ-B81 (DAR8) antigen concentrations were 500, 250, 125, 62.5, 31.25, 15.625 nM. The time was 60 seconds, and the antigen-antibody dissociation was carried out in the buffer for 60 seconds. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 seconds and repeated 3 times. The data were analyzed by DataAnalysis11 to obtain the affinity constant.

[0342] The measurement results of the affinity constants of C1q with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 19 below.

Table 37

[0343] 4.5 Detection of the ADCC activity of the anti-c-Met antibody and its ADC by Forte bio The ADCC effect means that effector immune cells with killing activity recognize the Fc segment of an antibody bound to a target cell antigen via the Fc receptor (FcR) expressed on their surface and directly kill the target cell.

[0344] The method for detecting the ADCC activity against MKN45 cells (from Sichuan Sibowo) expressing the c-MET antigen of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ is as follows. Weighed 0.5 g of BSA, added it to 50 mL of RPMI1640 basal medium, dissolved it completely, and then filtered it through a 0.22 μM micropore filter to obtain a sample diluent (RPMI1640 + 1% BSA) that could be used immediately. One day before the experiment, target cells (MKN45) in the logarithmic growth phase were digested with 0.25% trypsin in a biological safety cabinet, a single-cell suspension was prepared, counted with a cell counter, and the cell density was adjusted to 2×10 5 cells / mL with RPMI1640 + 10% FBS medium. Then, 100 μL / well was added to a cell culture plate and incubated overnight in a 37°C carbon dioxide cell incubator. The supernatant was discarded and stored. Effector cells (Jurkat-NFAT-CD16a, from Wuhan Taituozhong Biology) in the logarithmic growth phase were collected, counted with a cell counter, and the cell density was adjusted to 4×10 6 cells / mL with RPMI1640 basal medium. Then, 50 μL / well was added to the experimental wells containing MKN45 cells, namely, the sample group (ADC and antibody) and the negative control group (hIgG1). Daudi cells (CD20 target cells, from ATCC, catalog number CCL213) and effector cells (Jurkat-NFAT-CD16a) in the exponential growth phase were collected respectively, counted with a cell counter, appropriate amounts of Daudi cell and Jurkat-NFAT-CD16 cell suspensions were taken and mixed well, so that the final density of Daudi cells was 4×10 5 cells / mL and the final density of Jurkat-NFAT-CD16a cells was 4×10 6The cell density was adjusted with RPMI 1640 basal medium to be 1×10⁶ cells / mL, and 50 μL / well was added to the experimental wells of the positive control group of the experimental plate. Each test subject was diluted to a concentration of 2× (i.e., 40000 ng / mL) using the sample diluent, and further diluted to a total of 8 concentrations with a 5-fold serial gradient. Rituximab was diluted with a 7-fold gradient at a total of 8 concentrations. According to the layout of the experimental plate, 50 μL of the 2× test subject diluent diluted at each gradient was added to each well. The cell culture plate was placed on a microplate shaker and mixed well at 500 rpm / min for 5 minutes. The 96-well plate was placed in a 37 °C carbon dioxide constant temperature incubator and co-incubated for about 6 hours for culture. After the incubation, the Bio-Glory one-step (catalog number RA-GL04 from Adamas life), which had been thawed and equilibrated at room temperature in advance, was added to each experimental well of the cell culture plate, 50 μL / well, and shaken at room temperature for 10 minutes to mix well. The bioluminescence signal value was measured using a microplate reader.

[0345] The detection results of the ADCC activities of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ against MKN45 cells expressing the c-MET antigen are shown in Figure 17.

[0346] The results showed that in the co-culture system of MKN45 and JurkatNFAT-CD16a, at the same dose level, the ADCC activity induced by 45A5G10-HZ-B81 (DAR8) was significantly lower than that of 45A5G10-HZ.

[0347] The results showed that 45A5G10-HZ had a weak ADCC effect, while 45A5G10-HZ-B81 (DAR8) had no obvious ADCC effect.

[0348] 4.6 Detection of the CDC activity of the anti-c-Met antibody and its ADC by Forte bio The CDC effect means that when an antibody binds to the corresponding antigen on the cell membrane surface and simultaneously binds to complement C1q, it activates the typical complement-dependent cytotoxic effect pathway, forms a membrane attack complex, and thereby exerts a lytic effect on target cells.

[0349] The method for detecting the CDC activity against MKN45 cells expressing the c-MET antigens of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ is as follows: Weighed 0.5 g of BSA, added it to 50 mL of RPMI1640 basal medium and completely dissolved it, then filtered it through a 0.22 μM micropore filter to obtain a sample diluent (RPMI1640 + 1% BSA) that could be used immediately. Target cells (MKN45, Daudi) in the logarithmic growth phase were taken, prepared into a single cell suspension, counted with a cell counter, and the MKN45 cells were adjusted to 2×10 5 cells / mL using RPMI1640 basal medium, and the density of Daudi cells was adjusted to 6×10 5Adjusted to cells / mL, and after uniformly mixing by pipetting, 40 μL of the cell suspension was added to each well of the cell culture plate according to the layout of the experimental plate. One bottle of freeze-dried guinea pig serum complement (catalog number BM361Y from BERSEE) was taken and added to 1 mL of DMEM basal medium for re-dissolution. At this point, the complement concentration was 100%. 1000 μL of 100% complement was added to 1000 μL of DMEM basal medium and mixed well. At this point, the complement concentration was 50%. 20 μL of 50% serum complement was added to each experimental well of the cell culture plate to make the final concentration in the well 10%. Each test sample was diluted to a sample with a concentration of 2.5× (i.e., 250000 ng / mL) using the sample diluent, and further diluted to a total of 12 concentrations with a 3-fold serial dilution. 40 μL of the diluted solution of each test subject was added to each well. The cell culture plate was placed on a microplate shaker and mixed at 500 rpm / min for 5 minutes, and then placed in a carbon dioxide incubator at 37°C and incubated for about 6 hours. After the incubation, 50 μL of Cell Titer Turbo 2.0 reagent, which had been pre-melted and equilibrated at room temperature, was added to each experimental well of the cell culture plate, and shaken at room temperature for 10 minutes for thorough mixing. The bioluminescence signal value was measured with a microplate reader.

[0350] The detection results of the CDC activities of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ against MKN45 cells expressing the c-MET antigen are shown in Figure 18. The results showed that in the presence of complement, both 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ did not mediate the CDC effect at all dose levels.

[0351] ·4.7: Cross-detection of anti-c-MET antibody with human, monkey, rat, and mouse cMet Antigen proteins human c-MET (purchased from ACRO Biosystems, catalog number MET-H5227), cynomolgus c-MET (purchased from ACRO Biosystems, catalog number MET-C52H9), rat c-MET (purchased from Sino biological, catalog number 80004-R08H), mouse c-MET (purchased from Sino biological, catalog number 50622-M08H) were diluted with CBS at 1 μg / mL, and then the antigen was coded, blocked with 2% BSA (in PBS) at 37 °C for 1.5 hours, gradient-diluted 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ (starting from 1 μg / mL, 4-fold dilution, 8 concentration points) were added, incubated at 37 °C for 1 hour, HRP-labeled secondary antibody (Goat Anti-Human IgG, Monkey ads-HRP, purchased from southern biotech, catalog number F4522-V172E) was added, incubated at 37 °C for 1 hour, TMB substrate was added to develop color, and after terminating with the stop solution, the OD value of each well at a wavelength of 450 nm (reference wavelength 630 nm) was read mechanically.

[0352] The experimental results are shown in Table 20.

Table 38

[0353] Example 5. In Vivo Evaluation of Anti-Human cMet-ADC 5.1 Test of the Efficacy of Anti-Human cMet-ADC Against Non-Small Cell Lung Cancer Xenograft Tumors Test 1 To verify the in vivo efficacy evaluation of the anti-human cMet ADC drug and evaluate the antitumor effect of the test drug in a subcutaneous xenograft female Balb / c Nude mouse model, female Balb / c Nude mice (Tom Leehua) at 5-6 weeks of age were purchased. Human non-small cell lung cancer NCI-H358 cells that had grown to the logarithmic growth phase were digested with EDTA, resuspended in PBS, and 5×10 6 cells were subcutaneously inoculated into each mouse. Until the tumor grew to 100-200 m 3 , they were intravenously administered once a week at a dose of 1 mg / kg or 3 mg / kg each time.

[0354] The main observation indicators of this experiment are as follows. 1) TGI (%), the calculation formula is TGI (%) = (1 - T / C) × 100% (T and C are the relative tumor volumes at a specific time point in the treatment group and the control group respectively), and 2) the photos of the tumor volume and the tumor weight at the end of the test.

[0355] The experimental results are as shown in Table 21 and Figure 6. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) showed good tumor suppression effects in mice, and the tumor suppression effect was superior to that of ABT700-VcMMAE at a dose of 1 mg / kg. The change in the body weight of the mice is shown in Figure 7, indicating that the ADC did not affect the body weight of the mice throughout the administration process.

Table 39

[0356] Test 2 The purpose of this experiment is to evaluate the antitumor effect of the test article 45A5G10-HZ-B81 (DAR8) in a human non-small cell lung cancer cell NCI-H358 xenograft tumor model.

[0357] Female BALB / c Nu nude mice were subcutaneously inoculated with NCI-H358 cells, and when the tumors grew to approximately 180 mm 3 (200 mm 3 (as follows), 56 animals were screened and divided into 7 groups, namely the Vehicle group (physiological saline), the toxin (a small molecule toxin released by 45A5G10-HZ-B81 (DAR8), prepared with reference to Example A1.9 of WO2022170971) 0.07 mg / kg dosage group, the IgG1-B81 (isotype control antibody ADC, prepared with reference to IgG1-ADC-07 in Example 4.6.1 of WO2022170971) 3 mg / kg dosage group, and the 45A5G10-HZ-B81 (DAR8) 0.3, 1, and 3 mg / kg dosage groups, with 8 animals in each group. The animals in each group were administered once a week (QW) by tail vein injection for 3 consecutive weeks. The day of grouping was set as the first day of the test (Day1), and the administration started from Day1. After the final tumor test (22 days after the first administration, Day22), the animals were sacrificed, the tumors were separated, and weighed.

[0358] The results are shown in Table 22 and Figure 13. The tumor volumes of the animals in the 45A5G10-HZ-B81 (DAR8) 0.3, 1, and 3 mg / kg dosage groups were all significantly lower than those in the Vehicle group (P < 0.01 or P < 0.001), and the tumor growth inhibition rates (TGI) were 43.4%, 68.0%, and 81.1% respectively, showing a positive correlation with the dosage. The tumor volume of the animals in the IgG1-B81 3 mg / kg group was significantly lower than that in the Vehicle group (P < 0.01), and the tumor growth inhibition rate (TGI) was 49.3%. The results indicate that the anti-c-Met antibody-drug conjugate and its drug linker (toxin linker) provided by the present disclosure can achieve an anti-tumor effect without the need for either antibody endocytosis or antigen-positive expression of tumor cells.

[0359] The results of animal body weight measurement are shown in Figure 14. At the end of the test (Day22), in each dosage group of 45A5G10-HZ-B81 (DAR8), both the average body weight and the body weight change rate of the animals continued to increase during the administration period, and there was no significant difference (P > 0.05) compared with the Vehicle group.

Table 40

[0360] 5.2 Efficacy test of anti-human cMet-ADC against human colon cancer CDX model Using the same method, the antitumor effect of the anti-cMet ADC test drug was evaluated in a subcutaneous xenograft female BALB / c nude mouse animal model of the human colon cancer SW480 cell line.

[0361] The experimental results are shown in Table 23 and Figure 8. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) had good antitumor effects in mice against tumor cells with low expression of human cMet. The TGI at a dose of 5 mg / kg was significantly higher than that of ABT700-VcMMAE (DAR4). The change in mouse body weight is shown in Figure 9, indicating that the ADC did not affect the body weight of mice throughout the administration process. [Table 41]

[0362] 5.3 Efficacy test of anti-human cMet-ADC against human colorectal adenocarcinoma CDX model Using the same method, the antitumor effect of the cMet ADC test drug was evaluated in a subcutaneous xenograft female BALB / c nude mouse animal model of the human colorectal adenocarcinoma NCI-H716 cell line.

[0363] The experimental results are shown in Table 24 and Figure 10. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) showed excellent antitumor effects in mice. The TGI at a dose of 1 mg / kg was significantly higher than that of ABT700-VcMMAE (DAR4). The change in mouse body weight is shown in Figure 11, indicating that the ADC did not affect the body weight of mice throughout the administration process. [Table 42]

[0364] 5.4 Efficacy test of anti-human cMet-ADC against human colorectal cancer PDX model The purpose of this experiment is to evaluate the antitumor effect of the test article 45A5G10-HZ-B81 (DAR8) in the human colorectal cancer CR5088 PDX model (from Zhongmei Guanke Biotechnology Co., Ltd.).

[0365] NOD / SCID mice were subcutaneously inoculated with CR5088 tumor blocks (from Zhongmei Guanke Biotechnology Co., Ltd.) to establish a subcutaneous xenograft model of human colorectal cancer. The test was divided into a total of 4 groups, including 3 dose groups of the test drug 45A5G10-HZ-B81 (DAR8) (1 mg / kg, 3 mg / kg, and 10 mg / kg) and a vehicle control group, with 8 animals in each group. Administration was carried out once a week for a total of 3 weeks. The tumor inhibition rate (TGI) was calculated based on the tumor volume to evaluate the efficacy, and the safety was evaluated based on the body weight change and death status of the animals.

[0366] The results of the tumor volume are shown in Table 25 and Figure 15. 45A5G10-HZ-B81 (DAR8) showed a tendency to inhibit tumor growth at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, and the tumor inhibition rates were 41.54%, 95.89%, and 97.56% respectively. Here, the two dose groups of 3 mg / kg and 10 mg / kg had statistically significant differences compared with the blank control group, and the P values were both less than 0.001.

[0367] The results of the body weight change of the animals are shown in Figure 16. None of the mice in each group had significant weight loss (BWL < 15%), and there were no mice that died unexpectedly during the experiment, indicating good tolerance.

Table 43

[0368] 5.5 Efficacy test of anti-human cMet-ADC against transplanted gastric tumors The purpose of this experiment is to evaluate the antitumor effect of the test article 45A5G10-HZ-B81 (DAR8) in a human gastric cancer MKN45 xenograft tumor model.

[0369] Female NCG mice (Beijing Chuangmo) were subcutaneously inoculated with MKN45 cells (from JCBR JCRB0254) to establish a human gastric cancer model. The experiment was divided into a physiological saline control group, a toxin (toxin released by 45A5G10-HZ-B81 (DAR8), 0.23 mg / kg) treatment group, 45A5G10-HZ (10 mg / kg), IgG1-B81 (10 mg / kg) treatment groups, 45A5G10-HZ-B81 (DAR8) (1 mg / kg) treatment group, 45A5G10-HZ-B81 (DAR8) (3 mg / kg) treatment group, and 45A5G10-HZ-B81 (DAR8) (10 mg / kg) treatment group, with 8 animals in each group. Efficacy was evaluated based on the relative tumor growth inhibition rate (TGI), and safety was evaluated based on the body weight changes and mortality status of the animals.

[0370] The results of tumor volume are shown in Table 26 and Figure 19. 45A5G10-HZ has the effect of inhibiting the growth of human gastric cancer MKN45 subcutaneous xenograft tumors. 45A5G10-HZ-B81 (DAR8) was injected into the tail vein at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, once a week for 3 consecutive times, significantly inhibiting the growth of human gastric cancer MKN45 subcutaneous xenograft tumors. The relative tumor growth inhibition rates TGI were 72%, 96%, and 98% respectively, all showing statistically significant differences compared to the vehicle control group. The tumor volume of the animals in the IIgG1-B81 10 mg / kg group was significantly lower than that of the physiological group (P < 0.01), and the tumor growth inhibition rate (TGI) was 75%. It was shown that the anti-c-Met antibody-drug conjugate and its drug linker (toxin linker) provided by the present disclosure can achieve an antitumor effect without the need for either antibody endocytosis or antigen positive expression in tumor cells.

Table 44

[0371] Example 6 Plasma Stability Test of C-MET Antibody-Drug Conjugate The stability of 45A5G10-HZ-B81 (DAR8) in human plasma was evaluated by measuring the release of bioactive molecular toxins during incubation of 45A5G10-HZ-B81 (DAR8).

Table 45

[0372] 1. Experimental procedure Preparation of plasma: Rapidly thaw frozen plasma at 37 °C and place it on ice before use. Filter the thawed plasma through a 0.22 μm filter membrane and use it immediately after filtration.

[0373] Dilution process of the test drug: Step 1 Preparation of a 1 mg / mL drug dilution: Take a certain volume of 45A5G10-HZ-B81 (DAR8) with a concentration of 26.6 mg / mL, add the corresponding volume of 0.1 M PBS to prepare a 1 mg / mL drug dilution, filter it through a 0.22 μm filter membrane, and then use it.

[0374] Step 2 Preparation of stability samples: Take a certain volume of plasma or 0.1 M PBS, add the drug dilution to prepare a system with a concentration of 100 μg / mL, mix gently, dispense 200 μL into an EP tube (centrifuge tube) (this operation is carried out on ice at all time points), and seal it tightly.

[0375] 0 min sample: Immediately after dispensing the corresponding sample in Step 2, add 5 volumes of methanol, vortex for 2 minutes to mix, centrifuge for 10 minutes (4 °C, 17000×g), take out the supernatant, and store the supernatant at -60 °C or below until testing.

[0376] The stability samples prepared above were incubated at 37 °C for 24 h ± 10 min, 48 h ± 10 min, 72 h ± 15 min, 168 h ± 15 min, 240 h ± 15 min, 336 h ± 15 min, and 504 h ± 15 min, respectively. After the samples were precipitated with 5 volumes of methanol, the supernatant was frozen and stored at -60 °C or lower until testing. Free small molecules were detected using the LC-MS / MS method.

[0377] 2. Sample Detection The concentrations of small molecule drugs in all samples of each group were detected by the LC-MS / MS method.

[0378] 3. Data Processing Calculation of the theoretical total concentration of free small molecules: Ctotal (toxin) = (C administered concentration (45A5G10-HZ-B81 (DAR8)) / molecular weight 45A5G10-HZ-B81 (DAR8)) × antibody conjugation ratio (DAR) × molecular weight of the toxin Percentage of free small molecule drug release (%) = C (toxin) at each time point / Ctotal (toxin) × 100% The concentration data at each time point were retained with three significant digits, and the mean value, standard deviation, and release percentage were up to two decimal places.

[0379] 4. Results The summary of the concentrations and release percentages of the toxin in different species of plasma at each time point is shown in Table 27 below.

Table 46

[0380] Although specific embodiments of the present disclosure have been described in detail, those skilled in the art will be able to make various modifications and changes in detail based on all the teachings disclosed, and will understand that any of these changes are within the scope of the present disclosure. The scope of the present disclosure is given by the appended claims and any equivalents thereof.

Claims

Claim 1 An anti-c-MET antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the CDR of the heavy chain variable region and / or the CDR of the light chain variable region is identical to the CDR sequence of an antibody specified by the following sequences, or has 1, 2 or 3 amino acid substitutions compared to the CDR of an antibody specified by the following sequences, and the antibody specified by the following sequences is (1) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12, and / or (2) the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, an anti-c-MET antibody or an antigen-binding fragment thereof. Claim 2 The anti-C-MET antibody or an antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 10, and LCDR1, LCDR2, LCDR3 contained in the light chain variable region shown in SEQ ID NO: 9, HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 12, and LCDR1, LCDR2, LCDR3 contained in the light chain variable region shown in SEQ ID NO: 11, HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 2, and LCDR1, LCDR2, LCDR3 contained in the light chain variable region shown in SEQ ID NO: 1, HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 4, and LCDR1, LCDR2, LCDR3 contained in the light chain variable region shown in SEQ ID NO: 3, HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 6, and LCDR1, LCDR2, LCDR3 contained in the light chain variable region shown in SEQ ID NO: 5, or HCDR1, HCDR2, HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 8, and LCDR1, LCDR2, LCDR3 contained in the light chain variable region shown in SEQ ID NO: 7, and Preferably, the HCDR1-3 and the LCDR1-3 are determined according to the IMGT, Kabat or Chothia definition scheme, the anti-C-MET antibody or an antigen-binding fragment thereof according to Claim 1. Claim 3 An anti-C-MET antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, a. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 respectively. b. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 19 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 22 respectively. c. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 19 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 22 respectively. d. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 53 and SEQ ID NO: 19 respectively, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 22 respectively. e. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35 respectively. f. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively. g. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 32, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively. h. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 32, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively. i. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively. j. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 each containing the amino acid sequence shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 each containing the amino acid sequence shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively, or k. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3, which include the amino acid sequences shown in SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 44, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3, which include the amino acid sequences shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively. Preferably, a. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3, which include the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3, which include the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the IMGT definition scheme. b. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3, which include the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3, which include the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme. c. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3, which include the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3, which include the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme. d. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3, which include the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 53, and SEQ ID NO: 19, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3, which include the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Chothia definition scheme, e. The heavy-chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 respectively, and the light-chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 respectively, The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the IMGT definition scheme, f. The heavy-chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32 respectively, and the light-chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35 respectively, The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme, g. The heavy-chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 32 respectively, and the light-chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35 respectively, The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme, h. The heavy-chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 32 respectively, and the light-chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35 respectively, The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Chothia definition scheme, i. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47 respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the IMGT definition scheme. j. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50 respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme, or k. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 44 respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50 respectively. The above-mentioned HCDR1-3 and LCDR1-3 are determined according to the Chothia definition scheme, an anti-C-MET antibody or an antigen-binding fragment thereof.

4. The anti-C-MET antibody or an antigen-binding fragment thereof contains a heavy chain variable region and a light chain variable region, wherein 4, 3, 2, or 1 of the 6 CDRs of HCDR1-3 and LCDR1-3 contained in the heavy chain variable region and the light chain variable region are subjected to 1, 2, or 3 amino acid substitutions. Preferably, the substitution is a conservative substitution. The anti-C-MET antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The anti-C-MET antibody or an antigen-binding fragment thereof contains a heavy chain variable region and a light chain variable region, wherein (1) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9, (2) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 12, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 11, (3) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1, (4) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 3, (5) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 5, or, (6) The heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8, and / or the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:

7. The anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The anti-C-MET antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (1) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, (2) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, (3) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, (4) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, (5) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, or (6) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

7. The anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The anti-C-MET antibody or antigen-binding fragment thereof further comprises an antibody heavy chain constant region and a light chain constant region, Preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 or IgG4 constant regions, and the light chain constant region is selected from human antibody κ chain or λ chain constant regions. More preferably, the antibody is the anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a heavy chain constant region shown in SEQ ID NO: 51 and a light chain constant region shown in SEQ ID NO:

52.

8. The anti-C-MET antibody or antigen-binding fragment thereof comprises a or b, where a. A heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 56, and / or a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 57; Preferably, the anti-C-MET antibody comprises a heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 56 and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 57, or b. A heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 58, and / or a light chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 59; Preferably, the anti-C-MET antibody is the anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 58 and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO:

59.

9. An anti-C-MET antibody or antigen-binding fragment thereof, wherein the antibody competes with the anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 for binding to human C-MET.

10. A multispecific antibody, wherein the multispecific antibody comprises the anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and preferably, the multispecific antibody is a bispecific antibody, trispecific antibody or tetravalent antibody.

11. A nucleic acid molecule, wherein A nucleic acid molecule encoding an anti-C-MET antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, or a multispecific antibody according to claim 10.

12. A host cell, A host cell comprising the nucleic acid molecule according to claim 11.

13. An immune complex, Comprising an anti-c-MET antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 and an effector molecule that binds to the anti-c-MET antibody, Preferably, the effector molecule is selected from an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof. An immune complex.

14. A method used for in vivo and / or in vitro immunoassay or measurement of c-MET, The method comprising contacting the anti-c-MET antibody according to any one of claims 1 to 10 with a subject or a sample from a subject. A method used for immunoassay or measurement.

15. An antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, Containing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, or a multispecific antibody according to claim 10. An antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate.

16. The structure of the antibody-drug conjugate is represented by formula (I), 【Chemical 1】 wherein, Ab is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, or a multispecific antibody according to claim 10, D is an active drug unit, q is selected from integers from 1 to 20, for example, selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, L is a linking group that covalently binds to the antibody or its antigen-binding fragment Ab and the active drug unit D respectively, The L covalently binds to an amino residue or a sulfhydryl residue of the antibody Ab, Preferably, the L covalently binds to a sulfhydryl residue of the antibody Ab, more preferably, the L covalently binds to a sulfhydryl residue formed after the interchain disulfide bond of the antibody Ab is opened. Preferably, the active drug unit is selected from cytotoxic agents, more preferably, the active drug unit is selected from DNA topoisomerase inhibitors (e.g., camptothecin, DXD, camptothecin with modified substituents or DXD with modified substituents, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan and other camptothecin-based bioactive molecules), or microtubulin inhibitors (e.g., MMAF-type microtubulin inhibitors, MMAE-type microtubulin inhibitors), The antibody-drug conjugate according to claim 15, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, characterized in that.

17. L is a cleavable linker or a non-cleavable linker, Preferably, the cleavable linker contains peptide units, the peptide units contain 2 to 10 amino acid residues, and the amino acid residues are natural amino acid residues, unnatural amino acid residues, or amino acid residues shown in AA １ selected from the amino acid residues shown or stereoisomers thereof, More preferably, the peptide unit is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide containing at least one (e.g., one, two or three) AA １ amino acid residue represented by or a stereoisomer thereof, AA １ The structure of the amino acid residue shown in １ is as follows, [Chemical Formula 2] where, R ａ and R ｂ Of these, one is H and the other is 【Chemical Formula 3】 and r １ is 4, or R ａ is R ｂ together with the carbon atom to which they are commonly attached, forms a 5- to 6-membered heterocyclic ring substituted with R ０ and R ｍ１ and R ｎ１ are each independently selected from hydrogen, C １～６ alkyl group and C ３～６ cycloalkyl group, R ０ is selected from a C １～６ alkyl group, a C ３～６ cycloalkyl group, -NR ｍ２ R ｎ２ and optionally a 5- to 6-membered heterocyclyl group substituted with a C １～６ alkyl group, R ｍ２ and R ｎ２ are each independently selected from hydrogen and C １～６ alkyl groups, Preferably, the amino acid residue is selected from -Val-, -Ala-, -Gly-, -Cit-, -AA １ -, -Arg-, -Phe-, -Lys- and -Asn-; Preferably, the peptide unit is -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -valine-lysine- (-Val-Lys-), -valine-arginine- (-Val-Arg-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylalanine-lysine- (-Phe-Lys-), -phenylalanine-arginine- (-Phe-Arg-), -alanine-alanine-alanine- (-Ala-Ala-Ala-), -alanine-alanine-asparagine- (-Ala-Ala-Asn-), -valine-AA １ -glycine- (-Val-AA １ -Gly-), -valine-AA １ -alanine- (-Val-AA １ -Ala-), -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-) and -glycine-glycine-valine-alanine- (-Gly-Gly-Val-Ala-), the antibody-drug conjugate according to claim 16, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate.

18. said L is 【Chemical Formula 4】 and, where, L １ is 【Chemical Formula 5】 selected from, Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, C ６～１０ an aryl group, a 5- to 10-membered heteroaryl group, and an amide group (preferably selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond), Rx and Ry are independently selected from H and C １～４ an alkyl group, each m is independently selected from 0, 1, 2, 3, 4, 5, and 6, each y1 is selected from any integer from 1 to 6 (e.g., 4, 5, or 6), each y2 is independently selected from any integer from 0 to 15 (e.g., 6 to 15), each y3 is independently selected from 1, 2, and 3, each y4 is independently selected from 0 and 1, position 1 is linked to an antibody or an antigen-binding fragment thereof via an S atom, and position 2 is linked to L ２ or L ３ is linked to, L ２ does not exist or exists, and when L ２ exists, L ２ is ​ selected from, Each y1 is selected from any integer from 1 to 6 (for example, 4, 5, 6), each y2 is independently selected from any integer from 0 to 10 (for example, 6 to 10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, and position 1 is connected to L １ is connected to, position 2 is connected to L ３ is connected to, L ３ is [Chemical Formula 7] selected from, Position 1 is connected to L １ or L ２ and is connected, and position 2 is connected to L ４ or D L ４ does not exist or exists, and when L ４ exists, L ４ is [Chemical Formula 8] selected from, Position 1 is linked to L ３ The antibody-drug conjugate according to claim 16 or 17, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, wherein position 2 is linked to D.

19. L １ is 【Chemical Formula 9】 selected from, Position 1 is linked to Tb via an S atom, and position 2 is linked to L ２ or L ３ and is linked to Preferably, L １ is 【Chemical 10】 selected from, Position 1 is linked to Tb via an S atom, and position 2 is linked to L ２ or L ３ and L ２ does not exist or exists, and when L ２ exists, L ２ is 【Chemical 11】 selected from, Position 1 is connected to L １ and position 2 is connected to L ３ and Preferably, L ２ is absent, the antibody-drug conjugate according to claim 18, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate.

20. said 【Chemical Formula 12】 structure is shown as follows, 【Table 1】 Here, R １ and R ２ are independently selected from C １～６ alkyl groups and H, preferably C １～４ alkyl group, where position 1 is linked to the antibody or its antigen-binding fragment via an S atom, and position 2 is linked to D. The antibody-drug conjugate according to claim 19, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate.

21. The antibody-drug conjugate is a compound of formula (IIA-1), formula (IIA-2), formula (IIB-1) or formula (IIB-2), 【Chemical 13】 【Chemical 14】 【Chemical Formula 15】 【Chemical 16】 where, Ab is the antibody according to any one of claims 1 to 9 or its antigen-binding fragment, or the multispecific antibody according to claim 10, R １ and R ２ are independently selected from C １～６ alkyl groups and H, preferably C １～４ alkyl group, D is 【Chemical 17】 and, q is as defined in claim 16, preferably 2, 4, 6 or 8, The antibody-drug conjugate according to any one of claims 17 to 20, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate.

22. The antibody-drug conjugate has the following structure, 【Table 2-1】 【Table 2-2】 where, q is as defined in claim 16, preferably 2, 4, 6 or 8, Ab is the antibody according to any one of claims 1 to 9 or its antigen-binding fragment, or the multispecific antibody according to claim 10, Preferably, Ab is the antibody-drug conjugate according to claim 16, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, which comprises the heavy chain shown in SEQ ID NO: 56 and the light chain shown in SEQ ID NO: 57, or the heavy chain shown in SEQ ID NO: 58 and the light chain shown in SEQ ID NO:

59.

23. An antibody-drug conjugate group, comprising or consisting of the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate according to any one of claims 15 to 22, wherein the antibody-drug conjugate has one, two or more q values. Preferably, the average DAR of the antibody-drug conjugate group is selected from integers or decimals of 1 to 16, such as 1.5 to 2.5, 3.5 to 4.5, 5.5 to 6.5 or 7.5 to 8.

5. More preferably, the average DAR of the antibody-drug conjugate group is selected from about 2.0, 4.0, 6.0 or 8.

0. An antibody-drug conjugate group.

24. A pharmaceutical composition, comprising the antibody or its antigen-binding fragment according to any one of claims 1 to 9, the multispecific antibody according to claim 10, the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate according to any one of claims 15 to 22, the nucleic acid molecule according to claim 11, the immune complex according to claim 13 or the antibody-drug conjugate group according to claim 23, and optionally one or more pharmaceutical additives. A pharmaceutical composition.

25. Use in the preparation of a drug for the treatment or prevention of a disease related to the activity of C-MET of the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate according to any one of claims 15 to 22, the anti-C-MET antibody or its antigen-binding fragment according to any one of claims 1 to 9, the multispecific antibody according to claim 10, the nucleic acid molecule according to claim 11, the immune complex according to claim 13 or the antibody-drug conjugate group according to claim 23, or the pharmaceutical composition according to claim 24, Preferably, the disease related to the activity of C-MET is a tumor related to the activity of C-MET. Preferably, the tumor is selected from lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, or lung adenocarcinoma), colon cancer (e.g., human colon adenocarcinoma), rectal cancer, gastric cancer, colorectal cancer (e.g., colorectal adenocarcinoma) for use.

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