Anti-cMet antibody, antibody drug conjugate as well as preparation method and application of anti-cMet antibody and antibody drug conjugate

Abstract

The present disclosure provides an antibody or antigen binding fragment thereof targeting c-MET, an antibody drug conjugate and use thereof in the treatment of cancer. The disclosure also provides nucleotides encoding the c-MET antibody or the antigen-binding fragment thereof, a polynucleotide combination, an expression vector and an expression vector combination, a pharmaceutical composition comprising the c-MET antibody or the antigen-binding fragment thereof, an antibody drug conjugate, and applications thereof in the preparation of drugs for treating or preventing cancers.

Description

Anti-cMet antibodies, antibody-drug conjugates, their preparation methods and applications

[0001] This application is a divisional application of the patent application filed on December 22, 2023, with application number “202380088263.X” and title “Anti-cMet antibody, antibody-drug conjugate and preparation method and use thereof”. Technical Field

[0002] This application belongs to the field of pharmaceutical technology and relates to various antibodies, antibody-drug conjugates and their preparation methods, as well as their use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to their use in the prevention and / or treatment of tumor diseases. Background Technology

[0003] c-Met is a tyrosine kinase receptor expressed on the cell membrane. It binds to its ligand HGF via its Sema domain, triggering a downstream phosphorylation cascade that ultimately promotes cell proliferation. Currently, there are three main classes of inhibitors targeting c-Met kinase: HGF and c-Met antagonists, HGF and c-Met antibodies, and small-molecule c-Met inhibitors. Existing clinical results indicate that antibodies directly targeting HGF and c-Met, or small-molecule c-Met inhibitors, are not very effective.

[0004] Developing therapeutic antibodies against MET is challenging because antibodies that compete for HGF binding often lead to MET receptor dimerization and thus act as agonists (Prat M, et al. J Cell Sci 1998; 111 (Pt 2), 237-247). Onartuzumab was the first developed anti-c-Met antibody, derived from the humanization of the c-Met agonist antibody 5D5.

[0005] Chemotherapy using cytotoxic agents was once the standard treatment for cancer, but highly lethal cytotoxic molecules can damage normal cells, causing severe toxic side effects. Targeted antitumor drugs, possessing both targeting and antitumor activity, have become a hot topic in current cancer research; however, the selectivity of targeted drugs often leads to significant toxic side effects, thus limiting their therapeutic efficacy. Biological macromolecular drugs, such as antibodies or antibody fragments, while highly targeted, have limited or no therapeutic effect on solid tumors. Antibody-drug conjugates (ADCs), combining the targeting action of antibodies with the activity of bioactive molecules, act as a biological missile, offering promising advantages in efficacy and safety. Antibodies guide ADCs to bind to target cells, where they are subsequently internalized. The small molecule drug is then released intracellularly through enzymatic cleavage by specific enzymes, treating the disease.

[0006] Therefore, screening for a monoclonal antibody with high affinity and specific recognition of cMet, while effectively blocking HGF binding and downstream signaling pathways, is crucial for subsequent ADC drug development. For the C-Met target, developing differentiated, higher-quality, and safer antibodies or ADC drugs can provide cancer patients with a wider range of better treatment options and has broad market prospects. Summary of the Invention

[0007] This disclosure provides an antibody targeting c-MET or an antigen-binding fragment thereof, an antibody-drug conjugate, and its use in the treatment of cancer. This disclosure also provides nucleotides, combinations of polynucleotides, expression vectors and combinations of expression vectors encoding the aforementioned c-MET antibody or antigen-binding fragment thereof, pharmaceutical compositions comprising the aforementioned c-MET antibody or antigen-binding fragment thereof, antibody-drug conjugates, and their use in the preparation of medicaments for the treatment or prevention of cancer.

[0008] Firstly, this disclosure provides an antibody targeting c-MET or an antigen-binding fragment thereof; specifically, this disclosure provides an anti-c-MET antibody or an antigen-binding fragment thereof. In some embodiments, the c-MET is human c-MET.

[0009] In some embodiments, the antibody or its antigen-binding fragment comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3; and / or three complementarity-determining regions (LCDRs) 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 (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0010] In some embodiments, the CDR of the heavy chain variable region and / or the CDR of the light chain variable region are identical to or have 1, 2 or 3 amino acid substitutions compared to the CDR of an antibody defined by the following sequence: (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.

[0011] In some embodiments, the three complementarity-determining regions (HCDRs) from the heavy chain variable region disclosed herein, HCDR1, HCDR2, and HCDR3, are selected from: (i) the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 2, 4, 6, 8, 10, or 12, or (ii) sequences that, relative to any one of (i), contain at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions. Preferably, the HCDRs are determined according to the AbM, Chothia, Kabat, Contact, or IMGT definition scheme.

[0012] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region disclosed herein, LCDR1, LCDR2, and LCDR3, are selected from (i) the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 1, 3, 5, 7, 9, or 11, or (ii) sequences that, relative to any one of (i), contain at least one and no more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions. Preferably, the LCDRs are determined according to the AbM, Chothia, Kabat, Contact, or IMGT definition scheme.

[0013] In some embodiments, the anti-C-MET antibody or its antigen-binding fragment as described in any of the preceding embodiments 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 as described in any of the preceding embodiments 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 as described in any of the preceding embodiments 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 as described in any of the preceding embodiments 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 as described in any of the preceding embodiments 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 as described in any of the preceding embodiments 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 implementation schemes, HCDR1-3 and LCDR1-3 are identified based on the different measurement methods or systems or definition schemes used.

[0020] In certain embodiments, HCDR1-3 and LCDR1-3 are identified according to the AbM, Chothia, Kabat, Contact, or IMGT definition scheme. In certain embodiments, the complementarity determination regions HCDR1-3 and LCDR1-3 are determined based on the corresponding heavy and light chain variable regions according to the Chothia, Kabat, or IMGT definition scheme, as shown in the sequence and its specific information table.

[0021] In some embodiments, HCDR1, as defined by the IMGT, Kabat, or Chothia schemes, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:

[0022] In some embodiments, HCDR2, as defined by the IMGT, Kabat, or Chothia scheme, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:

[0023] In some embodiments, HCDR2, as defined by the IMGT, Kabat, or Chothia scheme, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 38 or 61, or comprises an amino acid sequence having one, two, or three alterations (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, HCDR3, as defined by the IMGT, Kabat, or Chothia scheme, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:

[0025] In some embodiments, LCDR1, as defined by the IMGT, Kabat, or Chothia scheme, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:

[0026] In some embodiments, LCDR2, as defined by the IMGT, Kabat, or Chothia scheme, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:

[0027] In some embodiments, LCDR3, as defined by the IMGT, Kabat, or Chothia scheme, comprises, or is composed of, the amino acid sequence shown in SEQ ID NO, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:

[0028] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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, which respectively comprise, or are composed 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, which respectively comprise, the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, or are composed of, the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the IMGT definition scheme.

[0029] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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, which respectively comprise the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:38 (QIRLKSLNYATHYAXSVKG, where X can be any amino acid, such as E or Q) and SEQ ID NO:19, or are composed of the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:38 (QIRLKSLNYATHYAXSVKG, where X can be any amino acid, such as E or Q) and SEQ ID NO:19; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, or are composed of the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, or are composed of the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:29. The amino acid sequence shown in NO:22; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0030] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: c. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:19; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, or are composed of, the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0031] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: d. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:19; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0032] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: e. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:53, and SEQ ID NO:19; the light chain variable region respectively comprises, or is composed of, the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Chothia definition scheme.

[0033] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: f. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the IMGT definition scheme.

[0034] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: g. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:61 (WIFPGSGNTKYX1X2KFX3G, wherein X1, X2, and X3 can 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 are composed of the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:61 (WIFPGSGNTKYX1X2KFX3G, wherein X1, X2, and X3 can 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; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:32 ... The amino acid sequences shown in NO:37 and SEQ ID NO:35, or the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:35, respectively; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0035] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: h. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35, or are composed of, the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0036] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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, which respectively comprise the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32, or are composed of the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35, or are composed of the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0037] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Chothia definition scheme.

[0038] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the IMGT definition scheme.

[0039] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: 1. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or are composed of, the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, or are composed of, the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme.

[0040] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: m. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise, or consist of, the amino acid sequences shown in SEQ ID NO:62, SEQ ID NO:60, and SEQ ID NO:44; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively comprise, the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, or consist of, the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; preferably, HCDR1-3 and LCDR1-3 as described above are determined according to the Chothia definition scheme.

[0041] In some embodiments, the anti-C-MET antibody or its antigen-binding fragment disclosed herein contains, compared to each of the aforementioned HCDR1-3, a total of no more than 5 amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions) in its three heavy chain CDRs. In some embodiments, the anti-C-MET antibody or its antigen-binding fragment disclosed herein contains, compared to each of the aforementioned LCDR1-3, a total of no more than 5 amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions) in its three light chain CDRs.

[0042] The anti-C-MET antibody or its antigen-binding fragment as described in any of the preceding claims comprises 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 replaced by 1, 2, or 3 amino acids. In some preferred embodiments, the replacement is a conservative replacement.

[0043] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments is a humanized antibody, a murine antibody, or a chimeric antibody. In some embodiments, the antibody disclosed herein is humanized. Humanization can be achieved by replacing one or more amino acid residues, particularly the framework region sequence, in the heavy chain and light chain variable regions of a non-human natural antibody with residues at corresponding positions in the variable regions of a conventional human antibody. Methods for humanizing antibodies are well known in the art. Typically, humanization substitutions are performed in a manner that preserves the favorable binding properties of the antibody. Assays for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art for identifying and selecting suitable mutations or combinations of humanized residues.

[0044] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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 disclosed herein (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 no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, or 12, preferably, the amino acid changes do not occur in the CDR region.

[0046] In some embodiments, the light chain variable region disclosed herein (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: 1, 3, 5, 7, 9, or 11; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, or 11; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, or 11, preferably, the amino acid changes do not occur in the CDR region.

[0047] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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: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.

[0048] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:10; and the light chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:9.

[0049] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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 as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:11.

[0051] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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 as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:2; and the light chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:1.

[0053] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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: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 as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:4; and the light chain variable region comprises or consists of an amino acid sequence as shown in SEQ ID NO:3.

[0055] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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: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 as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:6; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:5.

[0057] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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: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 as described in any of the preceding embodiments comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:8; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:7.

[0059] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments further comprises an antibody heavy chain constant region. In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments further comprises an antibody light chain constant region. In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments further comprises both an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is selected from the human IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, the light chain constant region is selected from the human antibody κ or λ chain constant region.

[0060] In some preferred embodiments, the antibody heavy chain constant region disclosed herein 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) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 51.

[0061] In some implementations, the amino acid change occurs in the Fc region.

[0062] In some embodiments, the constant region of the antibody light chain disclosed herein 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: 52; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 52; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) 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, the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising or consisting of an amino acid sequence as shown in SEQ ID NO:51, and the light chain constant region comprising or consisting of an amino acid sequence as shown in SEQ ID NO:52.

[0064] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments further comprises an antibody heavy chain. In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments further comprises an antibody light chain. In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments further comprises both an antibody heavy chain and a light chain.

[0065] In some preferred embodiments, the antibody heavy chain disclosed herein: (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 an amino acid sequence selected from SEQ ID NO: 56 or 58; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 56 or 58; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 56 or 58.

[0066] In some embodiments, the antibody light chain disclosed herein 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 an amino acid sequence selected from SEQ ID NO: 57 or 59; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 57 or 59; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 57 or 59.

[0067] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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 as described in any of the preceding embodiments comprises a heavy chain and a light chain, the heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:56, and the light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:57.

[0069] In some embodiments, the anti-C-MET antibody as described in any of the preceding embodiments 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 as described in any of the preceding embodiments comprises a heavy chain and a light chain, the heavy chain comprising or consisting of an amino acid sequence as shown in SEQ ID NO:58, and the light chain comprising or consisting of an amino acid sequence as shown in SEQ ID NO:59.

[0071] In some embodiments, this disclosure also provides an anti-C-MET antibody or an antigen-binding fragment thereof, wherein said antibody competitively binds to human C-MET with an anti-C-MET antibody as described in any of the preceding embodiments. As defined herein, an antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of said reference antibody to its antigen in a competitive assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of said antibody to its antigen in a competitive assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another; these assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays, bio-optical interferometry (e.g., Fortebio), or surface plasmon resonance (Biacore), etc.

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

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

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

[0075] In one embodiment of this disclosure, the amino acid alterations described herein include substitutions, insertions, or deletions of amino acids. In some embodiments, the amino acid alterations are conserved alterations. For a polypeptide sequence, a “conserved alteration” includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence.

[0076] Preferably, the amino acid changes described herein are amino acid substitutions, more preferably conservative substitutions. A conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, such as the substitution of one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid. For example, conservative substitutions often result in the substitution of a certain amino acid for a chemically similar amino acid. It is well known in the art to provide a table of conservative substitutions for functionally similar amino acids. The following lists eight groups of amino acids containing mutually conservative substitutions: 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); and 8) cysteine ​​(C), methionine (M). In some implementations, the term "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the antibody or binding protein molecule containing the amino acid sequence disclosed herein. For example, a conserved modified variant maintains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, binding affinity to the target antigen relative to the parent antibody or binding protein.

[0077] In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0078] In some embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant is modified (e.g., improved) relative to the parent antibody in certain biological properties (e.g., increased affinity) and / or will have some biological properties that are substantially retained by the parent antibody.

[0079] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0080] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein motifs known and readily available in the art. Suitable motifs for antibody derivatization include, but are not limited to, water-soluble polymers.

[0081] In some embodiments, the antibodies provided in this disclosure are multispecific antibodies, such as bispecific antibodies, trispecific antibodies, or tetraspecific antibodies.

[0082] Secondly, this disclosure provides a multispecific binding molecule, such as a multispecific antibody, comprising the aforementioned anti-C-MET antibody or its antigen-binding fragment. In some preferred embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0083] Thirdly, in some embodiments, this disclosure also provides a nucleic acid molecule that encodes an anti-C-MET antibody as described in any of the preceding claims, a fragment thereof, or a nucleic acid of any of its heavy or light chains.

[0084] For example, the nucleic acid disclosed herein comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 1-12 and 56-59, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1-12 and 56-59. As those skilled in the art will appreciate, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the molecule disclosed herein can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or by PCR amplification.

[0085] The heavy and / or light chains of the antibody molecules disclosed herein may be fused to the N-terminus with a secretory signal peptide and / or a tagged peptide that facilitates purification, for use in production and purification.

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

[0087] This disclosure also provides a host cell comprising a nucleic acid molecule or vector as described in any of the preceding embodiments. 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 suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as bacteria, e.g., *Escherichia coli*.

[0088] Polynucleotides encoding the polypeptide chain of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Once an expression vector containing one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

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

[0090] The antibodies prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A), size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0091] Fifthly, in some embodiments, this disclosure also provides an immunoconjugate (e.g., an antibody-drug conjugate) comprising an anti-C-MET antibody or its antigen-binding fragment as described in any of the preceding claims, and an effector molecule, wherein the effector molecule is conjugated to the anti-C-MET antibody or its antigen-binding fragment; preferably, the effector molecule is selected from radioisotopes, antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0092] In a sixth aspect, in some embodiments, this disclosure also provides methods for using the antibodies of the present invention or antigen-binding fragments thereof for diagnosis and detection, and compositions comprising the antibodies for diagnosis and detection.

[0093] In some implementations, any anti-c-MET antibody or its antigen-binding fragment provided herein can be used to detect the presence of c-MET in biological samples.

[0094] When used herein, the term "detection" includes both quantitative and qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some 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 implementation, an anti-c-MET antibody is provided for use in diagnostic or detection methods.

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

[0097] In some embodiments, a labeled antibody or fragment thereof is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0098] In some embodiments provided herein, the sample is obtained prior to treatment with the antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0099] In some implementations, c-MET is detected before treatment, for example, before the start of treatment or before a treatment after a treatment interval.

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

[0101] Therefore, in one embodiment, this disclosure also provides a method for immune detection or determination of C-MET, the method comprising the step of contacting a subject or a sample from a subject with an anti-C-MET antibody as described in any of the preceding claims.

[0102] Seventhly, this disclosure also provides an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, comprising the antibody or its antigen-binding fragment described in any of the preceding claims.

[0103] In some implementations, the antibody-drug conjugate structure is shown in formula (I): Formula (I) In which: Ab is the antibody or its antigen-binding fragment described in any of the preceding items; D is the active pharmaceutical unit; L is a linker, which is covalently linked to the antibody or its antigen-binding fragment Ab and the active pharmaceutical unit D, respectively; q is an integer selected from 1 to 20.

[0104] In some implementations, q is selected from 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 2-8, or 4-6; for example, it is an integer selected from 1-10.

[0105] Preferably, 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 is covalently linked to an amino or thiol residue on the antibody Ab; preferably, the L is covalently linked to a thiol residue on the antibody Ab; more preferably, the L is covalently linked to a thiol residue formed after the interchain disulfide bond on the antibody Ab is opened.

[0107] In some embodiments, L is a spliable connector or a non-spliable connector. Preferably, L is a spliable connector.

[0108] In some embodiments, the cleavable linker comprises a peptide unit containing 2-10 amino acid residues; the amino acid residues are selected from natural amino acid residues, non-natural amino acid residues, or amino acid residues represented by AA1 or their stereoisomers; in some embodiments, 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) amino acid residues represented by AA1 or their stereoisomers.

[0109] In some preferred embodiments, the peptide unit is a dipeptide, tripeptide, or tetrapeptide containing one amino acid residue represented by AA1 or its stereoisomer.

[0110] In some embodiments, the peptide unit is composed of the following amino acids: i. at least one (e.g., one, two, or three) amino acid residues represented by AA1 or their stereoisomers, and ii. at least one natural amino acid residue and / or at least one non-natural amino acid residue.

[0111] AA 1 The structures of the amino acid residues shown are as follows: AA 1 Where: R a R b In the middle, one is H, and the other is r 1 It is 4; or, R a With R b Together with the carbon atoms they are bonded to, they form R 0 Substituted 5-6 membered heterocycles; R m1 R n1 Each is independently selected from hydrogen and C. 1-6 Alkyl and C 3-6 cycloalkyl; R 0 Selected from C 1-6 Alkyl, C 3-6 cycloalkyl, -NR m2 R n2 and optional C 1-6 Alkyl-substituted 5-6 membered heterocyclic groups; R m2 R n2 Each is independently selected from hydrogen and C. 1-6 alkyl.

[0112] Preferably, the amino acid residues are selected from -Val-, -Ala-, -Gly-, -Cit-, and -AA. 1 -、-Arg-、-Phe-、-Lys-、and-Asn-.

[0113] Preferably, the peptide unit is selected from -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-).

[0114] In some preferred embodiments, L is L1 is selected from: , , , , , , , and Each Z is independently selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds, and C bonds. 6-10 Aryl, 5-10 membered heteroaryl and amide groups (preferably selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds); Rx and Ry are independently selected from H and C. 1-4 Alkyl group; each m is independently selected from 0, 1, 2, 3, 4, 5, and 6; y1 is selected from any integer between 1 and 6 (e.g., 4, 5, or 6); each y2 is independently selected from any integer between 0 and 15 (e.g., 6-15); each y3 is independently selected from 1, 2, and 3; each y4 is independently selected from 0 and 1; position 1 is linked to the antibody or its antigen-binding fragment via an S atom, and position 2 is linked to L2 or L3; in some embodiments, L1 is selected from... , , , , , , and Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0115] In some implementations, L1 is selected from , , , , , , , Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0116] In some implementations, L1 is selected from , , , , , and Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0117] In some implementations, L1 is selected from , and Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0118] In some implementations, L1 is selected from and Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0119] L2 may or may not exist; if L2 exists, L2 is selected from... , , , , , , and Each y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6-10), each y3 is independently selected from 1 or 2, and each y4 is independently selected from 0 or 1. One bit is connected to L1, and two bits are connected to L3. In some implementations, L2 may or may not exist. When L2 exists, L2 is selected from... , , , , and 1 bit is connected to L1, and 2 bits are connected to L3.

[0120] In some implementations, L2 may or may not exist; when L2 exists, L2 is selected from... , , , and 1 bit is connected to L1, and 2 bits are connected to L3.

[0121] In some implementations, L2 may or may not exist; when L2 exists, L2 is selected from... , , and 1 bit is connected to L1, and 2 bits are connected to L3.

[0122] In some implementations, L2 is not present.

[0123] In some implementations, L2 is selected from .

[0124] L3 is selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and One bit is connected to either L1 or L2, and two bits are connected to either L4 or D; L4 may or may not exist. If L4 exists, L4 is selected from... , , , , , , , , , and 1 bit is connected to L3, and 2 bits are connected to D.

[0125] In some preferred embodiments, the said The structure is as follows:

[0126] R1 and R2 are independently selected from C1-6 alkyl groups and H; preferably C1-6 alkyl groups. 1-4 Alkyl group; the 1-position is linked to the antibody or its antigen-binding fragment via an S atom, and the 2-position is linked to a D atom.

[0127] Preferably, the The structure is as follows:

[0128] In this configuration, position 1 is linked to the antibody or its antigen-binding fragment via the S atom, and position 2 is linked to the D atom.

[0129] In some embodiments, the active pharmaceutical unit is selected from cytotoxic agents. In some embodiments, the active pharmaceutical unit is selected from DNA topoisomerase inhibitors (e.g., camptothecin-based bioactive molecules, such as camptothecin, DXD, substituent-modified camptothecin or substituent-modified DXD, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetane, rubotecan) or microtubule inhibitors (e.g., MMAF-type microtubule inhibitors, MMAE-type microtubule inhibitors).

[0130] In some embodiments, the antibody-drug conjugate is a compound of formula (IIA-1) or formula (IIA-2): (IIA-1) (IIA-2) Wherein, Ab is the above-mentioned anti-c-Met antibody or its antigen-binding fragment, or a multispecific antibody; R1 and R2 are independently selected from C 1-6 Alkyl and H; preferably C 1-4 Alkyl; D is , , , , , , , or q is as defined above. In some preferred embodiments, q is 2, 4, 6, or 8.

[0131] In some embodiments, the antibody-drug conjugate is a compound of formula (IIB-1) or formula (IIB-2): (IIB-1) (IIB-2) Wherein, Ab is the above-mentioned anti-c-Met antibody or its antigen-binding fragment, for example, a multispecific antibody or its antigen-binding fragment; R1 and R2 are independently selected from C 1-6 Alkyl and H; preferably C 1-4 Alkyl; D is , , , , , , , or q is as defined above. In some preferred embodiments, q is 2, 4, 6, or 8.

[0132] In some embodiments, the antibody-drug conjugate has the following structure:

[0133] Ab and q are defined as above.

[0134] Eighthly, this disclosure provides a method for preparing an antibody-drug conjugate targeting C-MET, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers, or its pharmaceutically acceptable solvates, wherein the antibody-drug conjugate has the structure shown in Formula I, comprising the following steps: (1) reacting an anti-c-MET antibody or a fragment thereof with a reducing agent in a buffer solution to obtain a reduced antibody or a fragment thereof; preferably, the reducing agent is a disulfide bond reducing agent, such as TCEP; preferably, the buffer solution has a pH of 6.0-8.0, such as 6.5, 7.0, 7.5 or 8.0, more preferably a phosphate buffer solution; (2) crosslinking a drug linker (linker-drug conjugate) with the reduced antibody or a fragment thereof obtained in step (1) in a mixture of buffer solution and an organic solvent to obtain an antibody-drug conjugate targeting c-MET, wherein the buffer solution is as defined above, and preferably, the organic solvent is selected from dimethyl sulfoxide.

[0135] In some implementations of this disclosure, the c-MET antibody or fragment thereof is as defined above; the drug linker has a structure as shown in formula (IIIA-1), (IIIA-2), (IIIB-1), or (IIIB-2): , (IIIA-1) (IIIA-2) , (IIIB-1) (IIIB-2) where R1 and R2 are independently selected from C 1-6 Alkyl group, H; preferably C1-4 Alkyl; D is , , , , , , , or .

[0136] In some embodiments, the drug linker has the following structure:

[0138] The drug linker-payload disclosed herein can be prepared by various methods known in the art, such as chemical synthesis. The linker-payload in the above-mentioned antibody-drug conjugate can be prepared according to WO22022170971, and then conjugated with an antibody to form an ADC.

[0139] In the ninth aspect of this disclosure, this disclosure provides a group of antibody-drug conjugates comprising, or consisting of, the antibody-drug conjugates described in the seventh aspect, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates, wherein the antibody-drug conjugates have one, two, or more q values.

[0140] In some implementations, when a q-value of an antibody-drug conjugate in the antibody-drug conjugate group accounts for the majority (e.g., 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99%), the q-value and the average DAR are close.

[0141] In some implementations, when there is only one q-value antibody-drug conjugate in the antibody-drug conjugate group, the q-value and the average DAR are equal.

[0142] In some embodiments, when the antibody-drug conjugates of the antibody-drug conjugate group have two or more q values, the proportion of the antibody-drug conjugate with a particular q value among all antibody-drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0143] In some embodiments, the average drug-to-antibody ratio (average DAR) in the antibody-drug conjugate group is selected from an integer or decimal of 1-16, preferably 1-10.

[0144] In some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugate group is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5; in some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugate group is selected from about 2.0, 4.0, 6.0, or 8.0; in some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugates in the antibody-drug conjugate group 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 antibody-drug conjugate group contains ADCs with a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., drug loadings of 1.5, 2, 4, 6, and 8). It is noteworthy that degradation products can be generated, such that the mixture may also contain DARs of 1, 3, 5, and 7. Furthermore, the antibody-drug conjugate group may also have an average DAR greater than 8. The antibody-drug conjugates are produced by reduction and subsequent coupling of interchain disulfides. In some embodiments, the antibody-drug conjugates comprise both: antibody-drug conjugates with a DAR of 4 or lower (i.e., drug loadings of 4 or lower) and antibody-drug conjugates with a DAR of 6 or higher (i.e., drug loadings of 6 or higher).

[0146] In the tenth aspect of this disclosure, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment thereof as described in the first aspect, a multi-functional antibody as described in the second aspect, an antibody-drug conjugate or its stereoisomer as described in the seventh aspect, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, the nucleic acid described in the third aspect, an immunoconjugate as described in the fifth aspect or the antibody-drug conjugate group as described in the ninth aspect, and optionally one or more pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0147] In some embodiments, the pharmaceutical composition comprises effective amounts of the following substances: a first-aspect antibody or its antigen-binding fragment, a second-aspect multispecific antibody, a seventh-aspect drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, the third-aspect nucleic acid, the fifth-aspect immunoconjugate, or the ninth-aspect group of antibody-drug conjugates.

[0148] In some embodiments, the pharmaceutical composition comprises the above-described antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, and pharmaceutical excipients.

[0149] In some embodiments, the pharmaceutical compositions disclosed herein comprise the antibody-drug conjugate group of the ninth aspect, and pharmaceutical excipients.

[0150] In some embodiments, the pharmaceutical composition disclosed herein comprises the first aspect of the anti-c-MET antibody or its antigen-binding fragment thereof, and pharmaceutical excipients.

[0151] In some embodiments, the pharmaceutical compositions disclosed herein comprise the host cells disclosed herein, and pharmaceutically acceptable carriers and / or excipients, wherein the host cells comprise isolated nucleic acid molecules or carriers as described above.

[0152] In some embodiments, the pharmaceutical composition disclosed herein comprises a multispecific antibody as described in the second aspect of this disclosure, as well as pharmaceutical excipients.

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

[0154] In some embodiments, the drug-to-antibody ratio (mean DAR) in the pharmaceutical composition or antibody-drug conjugate group is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5; in some embodiments, the DAR in the pharmaceutical composition or antibody-drug conjugate group is selected from: 2±0.5, 4±0.5, 5±0.5, 6±0.5, 7±0.5, and 8±0.5; in some embodiments, the drug in the pharmaceutical composition or antibody-drug conjugate group... The ratio of drug to antibody (mean DAR) is selected from about 2.0, 4.0, 6.0, or 8.0; in some embodiments, the ratio of drug to antibody (mean DAR) in the pharmaceutical composition or antibody-drug conjugate group 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.

[0155] In some embodiments, the pharmaceutical composition comprises a first-aspect antibody or its antigen-binding fragment, a second-aspect multispecific antibody, or a seventh-aspect drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, and a buffer solution.

[0156] In some embodiments, the pharmaceutical composition comprises a seventh-faced drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof, and a buffer solution.

[0157] In some preferred embodiments, the buffer solution is selected from: histidine buffer, phosphate buffer. In some preferred embodiments, the buffer solution is selected from histidine buffer. In some preferred embodiments, the buffer solution is selected from 20 mM histidine buffer.

[0158] Eleventhly, the use of the antibodies or antigen-binding fragments thereof disclosed herein in the preparation of kits for detecting the presence or level of c-MET in a sample is provided. In another aspect, the disclosure provides diagnostic or therapeutic kits comprising one or more of the following substances: antibodies or antigen-binding fragments thereof disclosed herein, nucleic acids, vectors, host cells, multispecific antibodies, antibody-drug conjugates, groups of antibody-drug conjugates, or pharmaceutical compositions. Optionally, the diagnostic or therapeutic kits also include instructions for use. In some embodiments, the kits are suitable for the diagnostic or detection methods disclosed herein. In some embodiments, the kits are suitable for the therapeutic methods disclosed herein.

[0159] In a twelfth aspect, this disclosure provides the use of the substances disclosed herein (including the aforementioned antibody-drug conjugate compositions or the aforementioned pharmaceutical compositions) in the preparation of medicaments for the treatment and / or prevention of diseases related to abnormal cellular activity (e.g., cancer). In some embodiments, the antibody-drug conjugate composition or the aforementioned pharmaceutical composition is in an effective amount, such as a therapeutically effective amount.

[0160] In some embodiments, the use of the anti-C-MET antibody or its antigen-binding fragment, nucleic acid, vector, host cell or multispecific antibody disclosed herein in the preparation of a medicament for modulating (inhibiting or blocking) the activity of C-MET is provided.

[0161] In some embodiments, the use of the anti-C-MET antibody or its antigen-binding fragment, nucleic acid, vector, host cell antibody-drug conjugate or multispecific antibody disclosed herein in the preparation of a medicament for the treatment or prevention of diseases related to the activity of C-MET or to the target of C-MET is provided.

[0162] In some implementation examples, the use of the anti-C-MET antibody or its antigen-binding fragment, nucleic acid, vector, host cell, antibody-drug conjugate, or multispecific antibody disclosed herein in the preparation of a medicament for the treatment or prevention of tumors associated with C-MET activity is provided.

[0163] In some embodiments, the use of the substances disclosed herein in the preparation of a medicament for treating or preventing diseases related to the activity of C-MET or diseases related to the target of C-MET is provided, wherein the substances disclosed herein are selected from the antibody-drug conjugates disclosed herein, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates, the anti-C-MET antibody of the first aspect or its antigen-binding fragment, the multispecific antibody of the second aspect, the nucleic acid of the third aspect, the carrier of the fourth aspect, the carrier of the sixth aspect, the immunoconjugate of the seventh aspect, or the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0164] In some embodiments, the use of the antibody or antigen-binding fragment thereof of the first aspect of this disclosure, the multi-functional antibody of the second aspect, the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate of the seventh aspect, the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the preparation of a medicament for the treatment or prevention of diseases related to the activity of C-MET or diseases related to the target of C-MET is provided.

[0165] In a thirteenth aspect, this disclosure provides a method for treating and / or preventing diseases (e.g., tumors) associated with abnormal cellular activity, comprising the antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate of the seventh aspect, the nucleic acid of the third aspect, the carrier of the fourth aspect, the immunoconjugate of the fifth aspect, or the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0166] In aspects twelfth and thirteenth above, the diseases associated with C-MET activity, or with C-MET targets, or with abnormal cellular activity, include tumors, such as cancer. The cancer can be in its early, intermediate, or late stages, or metastatic. In some embodiments, the cancer can be a solid tumor or a hematologic malignancy.

[0167] In one embodiment, the tumor refers to an elevated level of c-MET protein (e.g., expression) or c-MET nucleic acid in an individual's tumor tissue or tumor cells, for example, compared to adjacent normal tissue or normal cells (e.g., normal cells in tissue) of the individual or the same tissue or cells of a healthy individual.

[0168] The tumors are selected from, but are 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, stomach cancer, and colorectal cancer (e.g., colorectal adenocarcinoma).

[0169] In the twelfth and thirteenth aspects above, the antibody or its antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate of the seventh aspect, the nucleic acid of the third aspect, the carrier of the fourth aspect, the immunoconjugate of the fifth aspect, or the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect may also be combined with other therapeutic methods or therapeutic agents for the treatment of related diseases or for related uses.

[0170] In some implementation schemes, the treatment is surgical or radiotherapy.

[0171] In a fourteenth aspect, this disclosure also provides pharmaceutical combinations or pharmaceutical combination products comprising the anti-c-MET antibody of the present invention or a fragment thereof (preferably an antigen-binding fragment), or an antibody-drug conjugate thereof, and one or more other therapeutic agents. Another object of the present invention is to provide a kit containing the pharmaceutical combination of the present invention, preferably said kit being in the form of a drug dosing unit. Dosing units can thus be provided according to a dosing regimen or drug administration interval.

[0172] In one embodiment, the kit of the present invention comprises, within the same package: - a first container containing a pharmaceutical composition comprising an anti-c-MET antibody or a fragment thereof; - a second container containing a pharmaceutical composition comprising other therapeutic agents.

[0173] In the fifteenth aspect, this disclosure also provides the use of the antibodies or antigen-binding fragments thereof of the first aspect and the multispecific antibodies of the second aspect for the preparation of antibody-drug conjugates.

[0174] In some embodiments, the antibody-drug conjugate is selected from antibody-drug conjugates of the seventh aspect, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates.

[0175] The immunoglobulin molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin. Preferably, the antibodies disclosed herein comprise or consist of a VH domain, VHCDR (often referred to herein as HCDR), VL domain, or VL CDR (often referred to herein as LCDR) having any of the amino acid sequences or fragments or variants thereof described in the Sequence and Specific Information Table.

[0176] Preferably, the antibody disclosed herein comprises or consists of a VH domain, a VH CDR (often referred to as HCDR herein), a VL domain, or a VL CDR (often referred to as LCDR herein) having any of the amino acid sequences or fragments or variants thereof described in the Sequence and Specific Information Table.

[0177] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the cluster are identical except for a small number of possible natural mutations. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a substantially homogeneous group of antibodies, and should not be construed as requiring special methods for preparation.

[0178] In some embodiments disclosed herein, monoclonal antibodies further specifically include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, while the remainder is identical or homologous to another type, class, or subclass of antibody, provided they possess the desired biological activity. Chimeric antibodies that can be used in this disclosure include primatized antibodies, which comprise a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

[0179] The term "antigen-binding fragment" refers to a portion of an antibody, preferably an antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dAb, and complementarity-determining region fragments, diabody, linear antibody, and single-chain antibody molecules. As used herein, the term "antigen-binding fragment" refers to a partial fragment of an antibody that has antigen-binding activity, wherein the fragment has complete or partial function of the antibody, including, but not limited to, single-chain Fv(scFv), Fab, Fab′, F(ab′)2, disulfide-linked Fv(sdFv), Fv, di-scFv, etc. The 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, the term is not limited to these molecules, as long as the fragment has binding affinity for the antigen. Furthermore, these functional fragments include not only fragments obtained by treating the full-length molecule of an antibody protein with a suitable enzyme, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0180] As used herein, the term "Fab'" refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above. However, Fab' disclosed herein also includes Fab' produced using genetically modified antibody genes.

[0181] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains, wherein the VL and VH are linked by a linker or directly (see, for example, 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, pp. 269-315 (1994)). Such scFv molecules may have a 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 with 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 this 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. In some cases, a disulfide bond may also exist between the VH and VL of the scFv. As used in this article, the term "di-scFv" refers to an antibody fragment formed by the linking of two scFvs.

[0182] The terms "variable region," "variable domain," or "variable structural domain" refer to the domains in the antibody heavy chain and / or light chain involved in antibody-antigen binding. Natural IgG antibodies VH and VL each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable structural domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable structural domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0183] The boundaries of the amino acid sequence of a CDR can be determined by various well-known definition schemes, such as the "Kabat" definition scheme (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, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various definition schemes is well known to those skilled in the art and is exemplified as shown below.

[0184] Relationship between CDR definition schemes

[0185] As used herein, the antibody or its antigen-binding fragment may contain variants, amino acid substitutions, deletions, or additions, but still retains the activity of binding the antigen.

[0186] The term "binding molecule" refers to any molecule capable of specifically binding to a target, such as an antibody or its antigen-binding fragment or fusion protein. The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule containing at least a first target-binding region and a second target-binding region, wherein 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 invention also encompass multispecific molecules containing multiple target-binding regions, such as trispecific binding molecules. In some embodiments, the multispecific binding molecules of the invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the invention are bispecific antibodies.

[0187] The term "bispecific antibody," also known as a "bifunctional antibody-drug conjugate," refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a conjugate arm. This conjugate retains the activity of each antibody, thus exhibiting both bifunctionality and bispecificity. In one embodiment, this document provides such a bispecific antibody, which has binding specificity against C-Met and binding specificity against a second antigen.

[0188] The term "multispecific antibody" includes, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies; the former are antibodies with binding specificity to three different antigens, while the latter are antibodies with binding specificity to four different antigens. In some embodiments, the multispecific antibody has binding specificity against c-Met and binding specificity against one or more other antigens.

[0189] The term "intact antibody" or "full-length antibody" refers to an antibody that contains an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be natural sequences (e.g., human natural constant region sequences) or amino acid sequence variants thereof. An intact antibody is preferably an intact antibody with one or more effector functions. In this disclosure, the "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimum amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which hypervariable region residues of human recipient immunoglobulins have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibodies) having the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with non-human residues. Furthermore, humanized antibodies may also contain residues not present in the recipient or donor antibody. These modifications are intended to further optimize antibody performance. Humanized antibodies typically contain at least one, usually two, variable regions, where all or almost all hypervanable loops correspond to those of non-human immunoglobulins, while the FRs are entirely or almost entirely sequences of human immunoglobulins. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc, typically human immunoglobulin Fc).

[0190] Intact antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant regions. The five main classes are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different antibody classes are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known in the art.

[0191] In this document, the CDR contained in the antibodies or antigen-binding fragments thereof disclosed herein can be determined according to various definition schemes known in the art. In some embodiments, the CDR contained in the antibodies or antigen-binding fragments thereof disclosed herein is preferably determined by the Kabat, Chothia, or AbM or IMGT definition schemes.

[0192] As used herein, the terms “framework residue region” or “FR residue” refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0193] The twenty common amino acids referred to in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this article, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala; arginine by R or Arg; glycine by G or Gly; and glutamine by Q or Gln.

[0194] As used herein, the term "immunoconjugate" refers to an effector molecule linked to an antibody or its antigen-binding fragment via a connector, such that the antibody or its antigen-binding fragment can act as a carrier to target and deliver the effector molecule to a target site. The term "effector molecule" refers to the active portion of the antibody or antibody fragment conjugated to this invention and may include any portion used for attaching 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 encompasses antibody-drug conjugates (ADCs). Suitable effector molecules or active portions linked to antibodies include, for example, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combinations thereof. In some embodiments, the immunoconjugate of this invention is an antibody-drug conjugate, i.e., an ADC.

[0195] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that inhibits or modulates (e.g., activates) an immune response. An immune response can be a humoral or cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0196] As used herein, the term “prevention” refers to methods implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., tumors and infectious diseases) in a subject. As used herein, the term “treatment” refers to methods implemented to achieve a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, “treatment” can also mean prolonged survival compared to expected survival (if no treatment was received).

[0197] As used herein, the term "subject" refers to a mammal, such as a primate, a non-human primate, or a human. In some embodiments, the subject (e.g., a human) has a tumor and an infectious disease, or is at risk of having such a disease.

[0198] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer and infectious diseases) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer and infectious diseases); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0199] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0200] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0201] The term "drug combination or combination product" refers to non-fixed combination products or fixed combination products, including but not limited to kits / reagents and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibodies of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the components produces an effect greater than that achieved by using any one ingredient alone in treating a disease or condition. The components may be in separate formulations, and their formulations may be the same or different.

[0202] The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0203] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0204] "Isolated" antibodies or molecules are those that have been separated from components of their natural environment. In some embodiments, the antibody or molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0205] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein, and if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. These variants may contain conserved changes.

[0206] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0207] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0208] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0209] The term "pharmaceutically acceptable" means that when the molecular bulk, molecular fragment, or composition is properly administered to an animal or human, it will not produce adverse, allergic, or other adverse reactions. Specific examples of substances that can serve as pharmaceutically acceptable carriers or components include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0210] The term “drug-to-antibody ratio” or “DAR” refers to the ratio of the amount of drug portion (D) to the amount of antibody portion (Ab) conjugated herein. The DAR of an ADC can range from 1 to 20, but higher loadings are possible depending on the number of binding sites on the antibody. The term DAR may be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. DAR can also be calculated as the average DAR of the molecular population in the product, i.e., the total ratio (molar ratio) of the drug portion (D) to the Ab portion conjugated herein, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC). This DAR is referred to herein as the average DAR. In some implementations, the average DAR value of the antibody-drug conjugates disclosed herein is 1.0-20.0, for example 1.0-18.0, 1.0-16.0, 2.0-14.0, 3.0-12.0, 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, 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, 7.8.0, 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, a range with two of these values ​​as endpoints.

[0211] During the mass spectrometry determination of DAR values, the antibody has been reduced to separate heavy and light chains. DAR1 represents a conjugate containing one toxin molecule coupled to either the light or heavy chain; DAR2 represents a conjugate containing two toxin molecules coupled to either the light or heavy chain; and DAR3 represents a conjugate containing three toxin molecules coupled to either the light or heavy chain.

[0212] The term "drug" refers to a chemical substance that can alter or reveal the physiological functions and pathological states of an organism, and can be used to prevent, diagnose, and treat diseases; in particular, it includes substances that inhibit or prevent cell function and / or cause cell death or damage. Drugs include cytotoxic agents, especially small molecule cytotoxic agents. There is no strict boundary between drugs and poisons. Poisons are chemical substances that produce harmful effects on the body and damage human health even in small doses; any drug, if taken in excessive amounts, can produce toxic reactions.

[0213] Cytotoxic agents are substances that inhibit or prevent cellular function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. Cytotoxic agents include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., Atmospheric iodine). 211 I 13 1. I 125 Y 90 Re 186 Re 188 、Sm 15 3. Bi 212 P 32 Radioactive isotopes of Lu), chemotherapy drugs, antibiotics, and ribolysins. Additionally, it includes, but is not limited to, DNA topoisomerase inhibitors (such as camptothecin-based bioactive molecules, such as camptothecin, DXD, substituent-modified camptothecin or substituent-modified DXD, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetane, rubotecan) or tubulin inhibitors (such as MMAF-type tubulin inhibitors, MMAE-type tubulin inhibitors).

[0214] To avoid ambiguity, the term "drug" that can be a component of an ADC does not refer only to "medicines" approved by pharmaceutical regulatory authorities, but also includes any compound with potential therapeutic biological activity in clinical trials, research, or academic studies. Furthermore, it should be understood that this differs from the meaning of "medicine" in the context of "preparation of a drug." It should be understood that drug molecules may require functionalization or derivatization for connection with a linker, and the resulting compounds are also included within the scope of drugs disclosed herein.

[0215] In this disclosure, the "active pharmaceutical unit" refers to the portion of an antibody-drug conjugate (ADC) other than the antibody and linker group, which is derived from the drug as defined above. For convenience, the "active pharmaceutical unit" in the ADC of this disclosure may be directly referred to by the name of the aforementioned drug, as understood by those skilled in the art.

[0216] As used in this disclosure, the term "pharmaceuticalally acceptable salt" refers to a salt that retains the biological effects and properties of the antibody-drug conjugates or drug-linked conjugates disclosed herein, and that such salt is not biologically or otherwise undesirable. The conjugates disclosed herein (including antibody-drug conjugates and drug-linked conjugates) may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In this disclosure, a pharmaceutically acceptable acid addition salt refers to a salt formed by the conjugates of this disclosure with an organic or inorganic acid, including but 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 refer to salts formed by the couplings in this disclosure with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by the couplings with N-containing organic bases.

[0217] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0218] As used in this disclosure, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans). Similarly, the compounds of this disclosure can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this disclosure covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0219] In this disclosure, solid lines (—), solid wedges, or dashed wedges may be used to depict the carbon-carbon bonds of the disclosed compounds. Using solid lines to depict bonds to asymmetric carbon atoms indicates that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) are included at that carbon atom. Using solid or dashed wedges to depict bonds to asymmetric carbon atoms indicates the presence of the indicated stereoisomers. Unless otherwise specified, the disclosed compounds are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-isomers, and mixtures thereof). The disclosed compounds may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0220] This disclosure also includes all pharmaceutically acceptable isotopic compounds identical to those disclosed herein, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds disclosed herein include (but are not limited to) isotopes of hydrogen (e.g., 2 H, 3 H); carbon isotopes (e.g., H); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes ... 32 P); and isotopes of sulfur (e.g. 35 S).

[0221] The compounds disclosed herein may exist as solvates (preferably hydrates), wherein the compounds contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the compound's crystal lattice. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0222] The scope of this disclosure also includes metabolites of the compounds disclosed herein, i.e., substances formed in the body upon administration of the compounds disclosed herein. Such products can be generated by, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc., of the administered compounds. Therefore, this disclosure includes metabolites of the compounds disclosed herein, including compounds prepared by methods that expose the compounds disclosed herein to mammals for a time sufficient to produce their metabolites.

[0223] This disclosure further includes, within its scope, prodrugs of the compounds disclosed herein. Typically, such prodrugs are functional group derivatives of the compounds that readily convert in vivo into the desired therapeutically active compound. Therefore, in these cases, the term "administration" used for the treatment methods of this disclosure should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but in which the prodrug form is converted in vivo into the aforementioned compound after administration to an individual. For example, conventional methods for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrug," ed. H. Bundgaard, Elsevier, 1985.

[0224] In this disclosure, pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a form, carrier, and improving stability, pharmaceutical excipients or excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local administration, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient or excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses. For information on the use and applications of pharmaceutical excipients, see also “Handbook of Pharmaceutical Excipients”, 8th edition, RCRowe, PJSeskey and SC Owen, Pharmaceutical Press, London, Chicago.

[0225] The pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the disclosed compound (including conjugates) or its pharmaceutically acceptable salt, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5-350 mg, and particularly preferably 1-250 mg. It should be noted that amounts exceeding the above ranges are sometimes permissible.

[0226] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media. The pharmaceutical composition can also be administered via intravenous infusion.

[0227] In this disclosure, regarding "L is linked to the antibody via a sulfur atom", those skilled in the art will understand that the sulfur atom comes from the thiol group contained in the antibody itself after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing it with the reducing agent TCEP to generate a thiol group -SH). In other words, the -S- between L and Ab is not an additional external sulfur atom.

[0228] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0229] In this disclosure, the term "linker group" or "linker" refers to a segment that links an active pharmaceutical unit (drug molecule) to an antibody portion. In this respect, a linker has functional groups that can form bonds with functional groups of the antibody or its antigen-binding fragment before being linked to the antibody (i.e., a linker precursor).

[0230] In this disclosure, the term "antibody-drug conjugate" or "ADC" refers to a substance in which an active pharmaceutical unit (drug molecule) is partially linked to an antibody or its antigen-binding fragment. In some embodiments of this disclosure, the active pharmaceutical unit and the target moiety are linked via a linker. This linker is cleavable under specific conditions (e.g., intracellular low pH) or under specific actions (e.g., lysosomal proteases), thereby separating a fragment of the bioactive compound (e.g., a c-Myc protein degrader) from the target moiety or the antibody or its antigen-binding fragment. In some embodiments of this disclosure, the linker comprises cleavable or cleavable units, such as peptides or disulfide bonds. In some embodiments of this disclosure, the active pharmaceutical unit and the target moiety or the antibody or its antigen-binding fragment are directly linked by a covalent bond, which is cleavable under specific conditions or actions, thereby separating the active pharmaceutical unit from the antibody or its antigen-binding fragment.

[0231] In this disclosure, the term "alkyl" refers to a straight-chain, fully branched hydrocarbon group that can be optionally substituted, preferably C1-C2. 10 More preferably, it is a C1-C8, C1-C6, or C1-C4 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-decyl, etc.

[0232] In this disclosure, the term "aryl" refers to a C6-C group having a monocyclic (e.g., phenyl) or fused ring (e.g., naphthyl, anthracene, phenanthryl, fluorene, etc.) that can be optionally substituted. 16 Aromatic hydrocarbon groups, preferably C6-C 10 Aromatic hydrocarbon groups.

[0233] In this disclosure, the term "heteroaryl" refers to a 5-16 nucleotide aromatic group containing one or more (e.g., 1, 2, 3 or 4) heteroatoms selected from N, O, S or P, which may be optionally substituted, preferably a 5-10 nucleotide aromatic group, more preferably a 5-6 nucleotide aromatic group. Examples of heteroaryl groups include imidazole, pyrazol, triazol, tetrazol, pyrrole, furanyl, thiophene, oxazol, isoxazol, thiazolyl, isothiazol, pyridinyl, pyrimidinyl, pyridazinyl, indole, azaindole (e.g., 7-azaindole), benzimidazole, benzopyrazol, benzofuranyl, benzothiophene, benzothiazolyl, dibenzofuranyl, dibenzothiophene, quinolinyl, isoquinolinyl, naphridinyl, carbazole, azacarbazole (e.g., 1-azacarbazole, 2-azacarbazole, 1,8-diazacarbazole), indoleazinyl, azaindoleazinyl, phenoxazinyl, phenthiazinyl, etc.

[0234] In this document, the terms "3-6 membered cycloalkyl" or "C" are used. 3-6"Cycloalkyl" refers to saturated cyclic alkyl groups containing 3-6 carbon atoms, including cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexyl.

[0235] In this document, the term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (of which at least one (e.g., 1, 2, or 3) ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom), including but not limited to pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, etc.

[0236] In this article, the term "5-6 membered heterocyclic group" refers to a cyclic group containing 5-6 ring atoms (of which at least one (e.g., 1, 2 or 3) ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom), including but not limited to pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazineyl, tetrahydropyranyl, etc.

[0237] The term "antibody-drug conjugate group" refers to a group or cluster of antibody-drug conjugates disclosed herein, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates, wherein the q of the antibody-drug conjugates may be the same or different. Alternatively, it may be referred to as an "antibody-drug conjugate mixture".

[0238] In some embodiments, the beneficial effects of the invention are that the antibody or antigen-binding fragment targeting c-MET developed herein binds to c-Met with high affinity, exhibiting high affinity for human and monkey cMet while also having good binding specificity, and does not bind to rat and mouse cMet.

[0239] In some implementations, the antibodies targeting c-MET or their antigen-binding fragments developed in this disclosure have antagonistic effects, antagonizing HGF-induced phosphorylation of c-Met and protein kinase B (PKB) rather than agonistic effects.

[0240] In some implementations, compared to control antibodies, such as ABT700, the antibodies or antigen-binding fragments of the c-MET targeted developed in this disclosure have highly efficient endocytic activity, higher affinity, different binding epitopes, and competitive c-Met binding activity with HGF ligands.

[0241] In some implementations, the anti-c-MET antibodies provided in this disclosure have extremely high levels of humanization and / or thermal stability, thereby enabling them to be druggable and safely administered to human subjects without triggering an immunogenic response.

[0242] Therefore, in some embodiments, the antibodies targeting c-MET or their antigen-binding fragments disclosed herein exhibit specificity, reduced toxicity, stability, and enhanced physical and functional properties compared to known therapeutic agents.

[0243] Meanwhile, compared to the development of c-MET signal blocking inhibitors, which only benefit a portion of the total number of cancer patients expressing c-MET, the c-MET ADC disclosed here can overcome some limitations of c-MET signal blocking inhibitors, enabling more cancer patients with low c-MET expression to benefit.

[0244] In some implementation schemes, the antibody-drug conjugates (ADCs) disclosed herein achieve enrichment of the tumor microenvironment, unique in vivo enzymatic cleavage characteristics of the linker, and conjugation mode with the target moiety, combined with extensive in vivo and in vitro efficacy screening and verification, to obtain a novel class of antibody bioactive molecular conjugates. The conjugates obtained using the above method can achieve a variety of surprising technical effects, including: in some embodiments, the conjugates obtained according to the above method have better solubility and excellent chemical stability, such as not undergoing the reversible Michael addition reaction caused by the maleimide linkage in traditional ADCs, thus achieving a high DAR value. In some embodiments, the DAR value of the conjugates can reach 6-8 or even higher; they have extremely high conjugation efficiency, in some embodiments, the conjugation efficiency can reach or exceed 90%; therefore, in one embodiment, this disclosure has discovered, through extensive research, a class of ADCs with high plasma stability, but which can also lyse in the tumor microenvironment (both inside and outside tumor cells), enabling release into both tumor cells and tumor tissues, maximizing the delivery of ADCs to tumor tissues and tumor cells, thereby also exerting therapeutic effects on tumors. Therefore, they can produce good anti-tumor effects in tumors with low or no antigen expression; the ADCs disclosed in this disclosure have shown significant tumor-suppressive activity on tumor cells with different c-MET expression levels.

[0245] In some embodiments, the ADC disclosed herein, after incubation at 37°C for 504 h, showed toxin release percentages of <0.6% in PBS solution, cynomolgus monkey plasma, and human plasma, demonstrating excellent plasma and circulatory stability. The conjugate (ADC) obtained according to the above method, through adjustments to the physicochemical properties of the linker and the overall ADC molecule, increases the exposure of the entire ADC molecule in a relatively acidic tumor environment. Therefore, the ADC exhibits better tumor tissue targeting, i.e., its enrichment capacity in the tumor microenvironment, increasing the intratumoral and blood concentration ratio of bioactive molecules and reducing the mechanism-related toxicity of the ADC molecule (the toxicity generated after ADC binds to and internalizes cell surface antigens in non-tumor tissues, also known as "on-target toxicity"), thus resulting in a higher therapeutic index. In some embodiments, the conjugate obtained according to the above method exhibits high stability in vivo circulation, reducing drug molecule shedding in non-target tissues and reducing "off-target" toxicity caused by toxin shedding in non-target tissues. In some embodiments, the bioactive molecules of the conjugate exhibit higher anti-tumor cell activity, thus demonstrating excellent by-stander effect. (Effect) ADCs can more effectively kill tumor cells with high antigen expression as well as tumor cells with low antigen expression or no antigen expression in tumor tissue. In some embodiments, the antibody-drug conjugates disclosed herein, utilizing the extracellular cleavage ability of their linkers in the tumor microenvironment, can form antibody-drug conjugates with antibodies that lack endocytosis ability. These antibody-drug conjugates still possess high anti-tumor activity. In some embodiments, the antibody-drug conjugates disclosed herein, utilizing the extracellular cleavage ability of their linkers in the tumor microenvironment and their enrichment ability in the tumor microenvironment, can form antibody-drug conjugates with antibodies that lack endocytosis ability and antibodies that lack the ability to bind to tumor cell extracellular antigens. These antibody-drug conjugates still possess high anti-tumor activity. In summary, the ADCs disclosed herein have significant clinical value.

[0246] This application also includes the following implementation schemes.

[0247] 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 are identical to or have 1, 2 or 3 amino acid substitutions compared to the CDR of an antibody defined by the following sequence: (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.

[0248] 2. The anti-C-MET antibody or its antigen-binding fragment as described in Embodiment 1, comprising: 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; 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; 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; 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; SEQ ID The HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in NO:6; and the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:5; or the HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:8; and the LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7; preferably, the HCDR1-3 and the LCDR1-3 are determined according to the IMGT, Kabat, or Chothia definition scheme.

[0249] 3. An anti-C-MET antibody or its antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: a. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain 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, which respectively contain the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; b. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:19; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; c. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:19; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; The amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:19; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; d. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:53, and SEQ ID NO:19; the light chain variable region respectively contains the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22; e. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; f. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35; g.The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35; h. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35; i. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:35. The amino acid sequence shown in NO:41; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; j. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; or k. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:62, SEQ ID NO:60, and SEQ ID NO:44; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which respectively contain the amino acid sequences shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. The amino acid sequence shown in NO:50; preferably, a. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the IMGT definition scheme; b.The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:19, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; c. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:18, and SEQ ID NO:19, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22, respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; d. The heavy chain variable region includes HCDR1, HCDR2, and LCDR3 as shown in SEQ ID NO:25, SEQ ID NO:25, and SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:19; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:22 respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Chothia definition scheme; e. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28 respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35 respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the IMGT definition scheme; f. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32 respectively; the light chain variable region includes LCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:36, SEQ ID NO:28, and SEQ ID NO:29 respectively; the heavy chain variable region includes LCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32 respectively; the light chain variable region includes LCDR1, HCDR2, and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO:28, and SEQ ID NO:29 respectively; the heavy chain variable region includes LCDR1, HCDR2, and LCDR3 as shown in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32 respectively; the light chain variable region includes LCDR1, HCDR2, and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO: LCDR1, LCDR2, and LCDR3 as shown in NO:37 and SEQ ID NO:35; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; g.The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:32, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35, respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; h. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:32, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:35, respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Chothia definition scheme; i. The heavy chain variable region includes HCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:32, respectively. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:41; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47 respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the IMGT definition scheme; j. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44 respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50 respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; or k. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:62, SEQ ID NO:60, and SEQ ID NO:44 respectively; the light chain variable region includes LCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50 respectively; HCDR1-3 and LCDR1-3 as described above are determined according to the Kabat definition scheme; or k. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:62, SEQ ID NO:60, and SEQ ID NO:44 respectively; the light chain variable region includes LCDR1, HCDR2, and LCDR3 as shown in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:40 respectively. LCDR1, LCDR2, and LCDR3 as shown in NO:49 and SEQ ID NO:50; HCDR1-3 and LCDR1-3 as described above are determined according to the Chothia definition scheme.

[0250] 4. The anti-C-MET antibody or its antigen-binding fragment according to any one of embodiments 1-3, comprising 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 replaced by 1, 2 or 3 amino acids; preferably, the replacement is a conservative replacement.

[0251] 5. The anti-C-MET antibody or its antigen-binding fragment according to any one of embodiments 1-4 comprises 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 ... NO:11 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; (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:2; NO:4 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, 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:3; 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:6; NO:5 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:8; 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:7, 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.

[0252] 6. The anti-C-MET antibody or its antigen-binding fragment according to any one of embodiments 1-5 comprises a heavy chain variable region and a light chain variable region, wherein: (1) the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:9; (2) the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; (3) the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:2; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:1; (4) the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:4; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:3; (5) the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:6; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; The amino acid sequence shown in NO:5 or composed of therefrom; or (6) the heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:8; and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:7.

[0253] 7. The anti-C-MET antibody or its antigen-binding fragment according to any one of embodiments 1-6, further comprising an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from the human IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from the human antibody κ or λ chain constant region; more preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO:51 and a light chain constant region as shown in SEQ ID NO:52.

[0254] 8. The anti-C-MET antibody or its antigen-binding fragment according to any one of embodiments 1-7 comprises: 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 composed of the amino acid sequence shown in SEQ ID NO:56 and a light chain containing or composed 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:57; NO:58 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:59, 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 comprises: a heavy chain containing or composed of the amino acid sequence shown in SEQ ID NO:58 and a light chain containing or composed of the amino acid sequence shown in SEQ ID NO:59.

[0255] 9. 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 or an antigen-binding fragment thereof as described in any one of embodiments 1-8.

[0256] 10. A multispecific antibody comprising the anti-C-MET antibody or its antigen-binding fragment as described in any one of embodiments 1-9, preferably, the multispecific antibody being a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0257] 11. A nucleic acid molecule encoding an anti-C-MET antibody or an antigen-binding fragment thereof as described in any one of embodiments 1-10, or a multispecific antibody as described in embodiment 10.

[0258] 12. A host cell comprising nucleic acid molecules as described in embodiment 11.

[0259] 13. An immunoconjugate comprising: an anti-c-MET antibody or an antigen-binding fragment thereof as described in any one of embodiments 1-9 and an effector molecule, wherein the effector molecule is conjugated to the anti-c-MET antibody; preferably, the effector molecule is selected from antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0260] 14. A method for in vivo and / or in vitro immunoassay or determination of c-MET, the method comprising the step of contacting an anti-c-MET antibody, as described in any one of embodiments 1-10, with a subject or a sample from the subject.

[0261] 15. An antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, comprising an antibody or its antigen-binding fragment as described in any one of embodiments 1-9, or a multispecific antibody as described in embodiment 10.

[0262] 16. The antibody-drug conjugate of embodiment 15, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, characterized in that the structure of the antibody-drug conjugate is as shown in formula (I): Formula (I) wherein: Ab is the antibody or its antigen-binding fragment as described in any one of embodiments 1-9, or the multispecific antibody as described in embodiment 10; D is the active pharmaceutical unit; q is an integer selected from 1-20, for example selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; L is a linking group, which is covalently linked to the antibody or its antigen-binding fragment Ab and the active pharmaceutical unit D, respectively; wherein L is covalently linked to an amino residue or a thiol residue on the antibody Ab; preferably, L is covalently linked to a thiol residue on the antibody Ab; more preferably, The active pharmaceutical unit is covalently linked to the thiol residue formed after the interchain disulfide bond on the L and antibody Ab is opened; preferably, the active pharmaceutical unit is selected from cytotoxic agents; more preferably, the active pharmaceutical unit is selected from DNA topoisomerase inhibitors (e.g., camptothecin-type bioactive molecules, such as camptothecin, DXD, substituent-modified camptothecin or substituent-modified DXD, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan) or microtubule inhibitors (e.g., MMAF-type microtubule inhibitors, MMAE-type microtubule inhibitors).

[0263] 17. The antibody-drug conjugate of embodiment 16, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein L is a cleavable linker or a non-cleavable linker, preferably, the cleavable linker comprises a peptide unit containing 2-10 amino acid residues selected from natural amino acid residues, non-natural amino acid residues, or amino acid residues represented by AA1, or their stereoisomers; 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) amino acid residues represented by AA1, or their stereoisomers. ； AA 1 The structures of the amino acid residues shown are as follows: AA 1 Where: R a R b In the middle, one is H, and the other is r 1 It is 4; or, R a With R b Together with the carbon atoms they are bonded to, they form R 0 Substituted 5-6 membered heterocycles; R m1 R n1 Each is independently selected from hydrogen and C. 1-6 Alkyl and C 3-6 cycloalkyl; R 0 Selected from C 1-6 Alkyl, C 3-6 cycloalkyl, -NR m2 R n2 and optional C 1-6 Alkyl-substituted 5-6 membered heterocyclic groups; R m2 R n2 Each is independently selected from hydrogen and C. 1-6 Alkyl group; preferably, the amino acid residue is selected from -Val-, -Ala-, -Gly-, -Cit-, -AA 1-, -Arg-, -Phe-, -Lys-, and -Asn-; preferably, the peptide unit is selected from -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-).

[0264] 18. The antibody-drug conjugate of embodiment 16 or 17, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein L is... L1 is selected from: , , , , , , , and Each Z is independently selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds, and C bonds. 6-10 Aryl, 5-10 membered heteroaryl and amide groups (preferably selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds); Rx and Ry are independently selected from H and C. 1-4 Alkyl group; each m is independently selected from 0, 1, 2, 3, 4, 5, and 6; y1 is selected from any integer between 1 and 6 (e.g., 4, 5, or 6); each y2 is independently selected from any integer between 0 and 15 (e.g., 6-15); each y3 is independently selected from 1, 2, and 3; each y4 is independently selected from 0 and 1; position 1 is linked to the antibody or its antigen-binding fragment via an S atom, and position 2 is linked to L2 or L3; L2 may or may not be present, and if present, L2 is selected from... , , , , , , and Each y1 is selected from any integer between 1 and 6 (e.g., 4, 5, 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6-10), each y3 is independently selected from 1 or 2, and each y4 is independently selected from 0 or 1. Bit 1 is connected to L1, and bit 2 is connected to L3; L3 is selected from... , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and One bit is connected to either L1 or L2, and two bits are connected to either L4 or D; L4 may or may not exist. If L4 exists, L4 is selected from... , , , , , , , , , and 1 bit is connected to L3, and 2 bits are connected to D.

[0265] 19. The antibody-drug conjugate of embodiment 18, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein L1 is selected from... , , , , , , and One position is connected to Tb via an S atom, and the second position is connected to L2 or L3; preferably, L1 is selected from... and Position 1 is connected to Tb via an S atom, and position 2 is connected to either L2 or L3; L2 may or may not exist. If L2 exists, L2 is selected from... , , , , and Bit 1 is connected to L1, and bit 2 is connected to L3; preferably, L2 does not exist.

[0266] 20. The antibody-drug conjugate of embodiment 19, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein, The structure is as follows:

[0267] Among them, R1 and R2 are independently selected from C 1-6 Alkyl and H; preferably C 1-4 Alkyl group; the 1-position is linked to the antibody or its antigen-binding fragment via an S atom, and the 2-position is linked to a D atom.

[0268] 21. The antibody-drug conjugate according to any one of embodiments 17-20, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the antibody-drug conjugate is a compound of formula (IIA-1), (IIA-2), (IIB-1), or (IIB-2): (IIA-1) (IIA-2) (IIB-1) (IIB-2) Wherein, Ab is the antibody or its antigen-binding fragment described in any one of embodiments 1-9, or the multispecific antibody described in embodiment 10; R1 and R2 are independently selected from C 1-6 Alkyl and H; preferably C 1-4 Alkyl; D is , , , , , , , or ;q is as defined in implementation scheme 16, preferably 2, 4, 6 or 8.

[0269] 22. The antibody-drug conjugate of embodiment 16, wherein its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, has the following structure:

[0270] Wherein, q is as defined in embodiment 16; preferably 2, 4, 6 or 8; Ab is the antibody or its antigen-binding fragment described in any one of embodiments 1-9, or the multispecific antibody described in embodiment 10; preferably, Ab comprises: a heavy chain as shown in SEQ ID NO:56 and a light chain as shown in SEQ ID NO:57, or a heavy chain as shown in SEQ ID NO:58 and a light chain as shown in SEQ ID NO:59.

[0271] 23. A group of antibody-drug conjugates comprising, or consisting of, any one of the antibody-drug conjugates described in embodiments 15-22, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates, wherein the antibody-drug conjugates have one, two, or more q values; preferably, the average DAR of the group of antibody-drug conjugates is selected from an integer or decimal number from 1 to 16, for example, selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5; more preferably, the average DAR of the group of antibody-drug conjugates is selected from about 2.0, 4.0, 6.0, or 8.0.

[0272] 24. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of embodiments 1-9, a multispecific antibody as described in embodiment 10, an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate as described in any one of embodiments 15-22, a nucleic acid molecule as described in embodiment 11, an immunoconjugate as described in embodiment 13, or a group of antibody-drug conjugates as described in embodiment 23, and optionally one or more pharmaceutical excipients.

[0273] 25. Use in the preparation of a medicament using an antibody-drug conjugate according to any one of embodiments 15-22, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof; an anti-C-MET antibody according to any one of embodiments 1-9 or its antigen-binding fragment thereof; a multispecific antibody according to embodiment 10; a nucleic acid molecule according to embodiment 11; an immunoconjugate according to embodiment 13 or a group of antibody-drug conjugates according to embodiment 23; or a pharmaceutical composition according to embodiment 24, wherein the medicament is used to treat or prevent diseases related to C-MET activity; preferably, the disease related to C-MET activity is a tumor related to C-MET activity; 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). Attached Figure Description

[0274] Figure 1. Cross-detection of humanized antibody with rat cMet; Figure 2. Cross-detection of humanized antibody with mouse cMet; Figure 3. Competition detection of humanized antibody with ABT700 epitope; Figure 4. Humanized antibody blocks HGF-induced ERK phosphorylation; Figure 5. Humanized antibody induces cMet protein degradation; Figure 6. In vivo efficacy of anti-human cMet ADC in NCI-H358 CDX model (Test 1); Figure 7. Changes in mouse body weight during administration of anti-human cMet ADC in NCI-H358 CDX model (Test 1); Figure 8. In vivo efficacy of anti-human cMet ADC in SW480 CDX model; Figure 9. Changes in mouse body weight during administration of anti-human cMet ADC in SW480 CDX model; Figure 10. In vivo efficacy of anti-human cMet ADC in NCI-H716 CDX model; Figure 11. In vivo efficacy of anti-human cMet ADC in NCI-H716 CDX model. Figure 12. Validation of c-Met target expression levels in different tumor cells; Figure 13. In vivo efficacy of anti-human cMet ADC in the NCI-H358 CDX model (Test 2); Figure 14. In vivo efficacy of anti-human cMet ADC in the NCI-H358 CDX model (Test 2); Figure 15. In vivo efficacy of anti-human c-Met ADC in the CR5088 PDX model; Figure 16. In vivo efficacy of anti-human cMet ADC in the CR5088 PDX model; Figure 17. ADCC activity test of anti-human cMet antibody and ADC on MKN45 cells, the upper figure is the test curve of positive control, and the lower figure is the test curve of 45A5G10-Hz antibody and its ADC; Figure 18. Anti-human cMet antibody and ADC The CDC activity assay on MKN45 cells is shown in Figure 19. The upper figure shows the test curve for the positive control, and the lower figure shows the test curve for the 45A5G10-Hz antibody and its ADC. Detailed Implementation

[0275] The following description of specific embodiments further illustrates this disclosure, but it is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this disclosure without departing from its fundamental ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0276] Sequence and its specific information:

[0277] This disclosure is now described with reference to the following examples which are intended to illustrate (and not limit) this disclosure.

[0278] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this disclosure are substantially based on those 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., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples described herein are by way of illustration and are not intended to limit the scope of protection claimed herein.

[0279] The abbreviations used in this disclosure have the following meanings, and abbreviations for which no meaning is provided have the meaning commonly understood in the art:

[0280] Example 1, Screening and preparation of anti-c-Met antibodies 1.1 Antigen information 1) Plasmid: Human cMet cDNA ORF Clone was purchased from Sinocare (product number HG10463-CH). Then, the nucleotide sequence corresponding to the extracellular segment of human cMet protein 25-932 amino acids was cloned into pTT5-mFc and pTT5-hFc vectors (from Sichuan Sibowo Biotechnology Co., Ltd.), and finally expression plasmids fused with human IgG Fc segment and mouse IgG Fc segment were formed respectively.

[0281] 2) Antigen expression: HEK293E cells (from Sichuan Sibowo Biotechnology Co., Ltd.) were transiently transfected with PEImax (Polysciences, 24765-1). After 7 days of expression, hcMet-ECD-mFc and hcMet-ECD-hFc antigen proteins were purified by ProA packing material (GE, Mabselect XL).

[0282] 3) Commercialized antigen proteins: Human cMet extracellular domain recombinant protein (Human HGF R / c-MET Protein, FcTag) is from Bipsys (Catalog No.: MET-H5256), Human cMet extracellular domain recombinant protein (Human HGF R / c-METProtein, His Tag) is from Bipsys (Catalog No.: MET-H5227), Monkey cMet extracellular domain recombinant protein (c-METProtein, Cynomolgus, Rhesus, Recombinant (His Tag)) is from Sinocare (Catalog No.: 90304-C08H), Rat cMet extracellular domain recombinant protein (Rat-cMet-hFc, c-MET Protein, Rat, Recombinant (hFc Tag)) is from Sinocare (Catalog No.: 80004-R02H), and Mouse cMet extracellular domain recombinant protein (mouse cMet-ECD-His) is from Sinocare (Catalog No.: 80004-R02H). c-MET Protein, Mouse, Recombinant (ECD, His Tag) was from Sinocare (Catalog No.: 50622-M08H), and human HGF recombinant protein (HGF Protein, Human, Recombinant) was from Sinocare (Catalog No.: 10463-HNAS).

[0283] 1.2 Immunization and Antibody Screening Four female mice from each of the CD1 (Vitaliva), KM (Vitaliva), and Balb / c (Jicui Yaokang) strains were enrolled at approximately 6 weeks of age. Immunization was performed using cMET-His and hcMET-mFc (Sichuan Sibowo Biotechnology Co., Ltd.), with two booster immunizations. Serum titers were measured by ELISA. For mice with high ELISA protein titers, the affinity titer of tumor cells for MKN-45 and the competitive FACS titer of MKN45-HGF were assessed using FACS. Finally, spleen and lymph nodes from one mouse each of the CD1, KM, and Balb / c strains were used to prepare cell suspensions, which were then fused with SP2 / 0 mouse myeloma cells for hybridoma fusion. Primary screening was performed using human hMET-ECD-hFc antigen protein via ELISA, and positive hybridoma clones were selected. Hybridoma cells were cultured in serum-free medium, and the collected monoclonal supernatant was purified using ProA packing material to obtain mouse antibodies. The affinity of murine antibodies for human MET-ECD-hFc and cynomolgus monkey MET-ECD-hFc proteins was detected by ELISA. The affinity of murine antibodies for MKN45 tumor cells and their ability to block competition with MKN45-HGF ligands were detected by FACS. After multiple rounds of screening, a total of 18 positive clones were obtained. The 18 murine antibodies were evaluated, and the evaluation results of the four clones with high affinity are shown in Table 1.

[0284] Table 1. Detection of affinity and HGF competitiveness of anti-cMET mouse antibodies

[0285] ND indicates that the hook sequences for the four monoclonal antibodies with high affinity, 55A10G6, 45A5G10, 51D5B2, and 44H10E8, were not detected. The hook sequence method was as follows: approximately 1 × 10⁻⁶ candidate hybridoma cells were collected. 5 RNA was extracted using Trizol, and cDNA was obtained by reverse transcription using the PrimeScript RTreagent kit via PolyA. Upstream primers were designed for the heavy and light chains, and downstream primers were designed for the CH1 region of the heavy chain and the CL region of the light chain. The PCR amplification products were then recovered using an agarose gel extraction kit. The samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The specific sequences of the murine antibody variable region and CDR are shown in the sequence information table.

[0286] 1.3 Antibody humanization employed the CDR grafting method. First, the human germline sequence with the highest homology to the original mouse sequence was identified using conventional BLAST and used as a template. The CDR of the mouse antibody was then grafted onto the human template to construct a chimera. Based on structural analysis, the FR amino acids in the mouse antibody that retained their original conformation were identified, and the corresponding amino acids in the chimera were reverted to mouse amino acids to maintain the original affinity. The constructed humanized antibody underwent calculations and immunogenicity analysis to identify high-immunogenic fragments and replace low-immunogenic fragments. For the replacement of high-immunogenic sites in the CDR, such as the replacement of 45A5G10 Kabat HCDR2 in humanization, from QIRLKSLNYATHYA... E SVKG (SEQ ID No. 17) was replaced with QIRLKSLNYATHYA Q SVKG (SEQ ID No. 18) replaces 55A10G6 Kabat HCDR2 from WIFPGSGNTKY IE KF K G (SEQ ID No. 30) replaced with WIFPGSGNTKY SQ KF QG (SEQ ID No. 31). Humanization of murine antibodies 55A10G6 (abbreviated as 55A10G6) and 45A5G10 (abbreviated as 45A5G10) yielded 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 shown in the sequence information table, where the CDRs are provided according to the IMGT, chothia, and Kabat definitions.

[0287] 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 resulting sequences were submitted to Universal Biotechnology for gene synthesis. After codon optimization, the sequences were constructed into the PTT5 vector. After plasmid synthesis, HEK293E cells (from Sichuan Sibowo Biotechnology Co., Ltd.) were transfected with PEImax. Expression was carried out for approximately 7 days, and the supernatant was collected by centrifugation. The supernatant was purified using ProA packing material. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and the cells were stored at -20°C. Antibody 55A10G6-hz was obtained.

[0288] 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 resulting sequences were submitted to Universal Biotech for gene synthesis. After codon optimization, the sequences were constructed into the PTT5 vector. After plasmid synthesis, HEK293E cells were transfected using PEImax and expressed for approximately 7 days. The supernatant was collected by centrifugation. The supernatant was purified using ProA packing material. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and the cells were stored at -20°C. Antibody 45A5G10-hz was obtained.

[0289] The amino acid sequences of the heavy and light variable regions of the control antibody (ABT700, sequence from KEGG, ID: D11307) were submitted to Universal Biotech for gene synthesis. After codon optimization, the antibody was constructed into the PTT5 vector. Once the plasmid was synthesized, it was transfected into HEK293E cells via PEImax. After approximately 7 days of expression, the supernatant was collected by centrifugation. The supernatant was purified using ProA packing material. All purified antibody was ultrafiltered into PBS buffer, the concentration was determined, and the antibody was stored at -20°C.

[0290] Example 2, Evaluation of anti-c-Met antibodies 2.1 Evaluation of anti-c-Met antibody protein ELISA affinity: The antigen protein cMet-ECD-his was diluted 1 μg / ml with carbonate buffer (CBS), coated with the antigen, and blocked with 2% BSA (in phosphate-buffered saline PBS) at 37°C for 2 hours. Serially diluted humanized antibody (2 μg / mL starting, 3-fold dilution, 11 concentration points) was added, and incubated at 37°C for 2 hours. HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164) was added, and incubated at 37°C for 1 hour. TMB substrate was added for color development, and the reaction was stopped with 2M HCl. The absorbance was read at 450 nM. The antigen protein anti-His-Rabbit Fc (from Chengdu Apak Biotechnology Co., Ltd.) was diluted 1 μg / ml with CBS, coated with the antigen, blocked with 2% BSA (in PBS) at 37°C for 2 hours, and 0.5 μg / ml of the antibody was added. Incubate the cyno.cMet-ECD-His sample at 37°C for 2 hours. After 2 hours, add serially diluted humanized antibody (2 ug / mL as the starting point, 3-fold dilution, 11 concentration points). Incubate at 37°C for 2 hours. Add HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164). Incubate at 37°C for 1 hour. Add TMB substrate for color development. Stop the reaction with 2M HCl and read the absorbance at 450 nM.

[0291] The experimental results are shown in Table 2. Both humanized antibodies showed high affinity for both human and cynomolgus monkey Met proteins, and the affinity for cynomolgus monkey Met was significantly stronger than that for ABT700.

[0292] Table 2. Affinity evaluation of humanized antibody ELISA

[0293] 2.3 Flow Cytometry Affinity Evaluation of Anti-c-Met Antibody: Trypsin digestion and centrifugation were used to collect MKN45 cells (Nanjing Kebai, CBP60488). Cells were washed three times with pre-cooled PBS and resuspended in 1% BSA (in PBS) at 2^10 cells per well. 5 50 μL of cells were seeded into 96-well conical plates. The anti-cMet humanized antibody to be tested was serially diluted with 1% BSA, starting at 20 μg / ml, with 4-fold dilutions and 8 dilution points. 50 μL of the diluted antibody was mixed with the cells in the conical plate and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 100 μL of 1% BSA (containing 1 μL of anti-human APC fluorescent secondary antibody, BioLegend, catalog number 410712) was added to each well and incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and detected by flow cytometry (Beckman, Cytoflex).

[0294] The experimental results are shown in Table 3. Both humanized antibodies showed high affinity for MKN45 cells, and the affinity of both antibodies for MKN45 cells was higher than that of the control antibody ABT700.

[0295] Table 3. Flow cytometry affinity evaluation of humanized antibodies

[0296] 2.4 Evaluation of Dynamic Affinity of Anti-c-Met Antibodies The dynamic affinity of humanized antibodies was determined using ForteBio. The experimental steps were as follows: 1. Sensor Preparation: Take out the ProA sensor and pre-wet it with PBST dilution buffer (pH 7.4) for 10 min; 2. Sample Dilution: Dilute the antibodies to be solidified to 5 ug / ml. The antigen h.cMet-ECD-His (Bepsys, catalog number: MET-H5227) was started at 500 nM, 2-fold diluted, with 5 concentration points, and a 0 concentration point was set; 3. Program Setup: Place the sensor plate and sample plate, start the program, and regenerate the sensor with 20 mM glycine solution (pH 1.7); 4. Data Analysis: Analyze the data using Octet analysis software. The experimental results are shown in Table 4. Antibodies 45A5G10-Hz and 55A10G6-Hz showed high dynamic affinity to human cMet protein.

[0297] Table 4. Dynamic affinity assay of humanized antibodies

[0298] 2.5 Quality Identification of Anti-c-Met Antibodies 1) The purity of the humanized antibody was determined by SEC. The detection method is as follows: Instrument: Waters Alliance e2695 HPLC; Column: Thermo MabPac SEC-1, 5 μm, 7.8 300 mm; Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA; Instrument parameters: Sample chamber temperature: 8℃; Column temperature: 30℃; Flow rate: 0.5 ml / min; Injection volume: 20 ug; Detection wavelength: 280 nm; Isocratic run: 30 min.

[0299] 2) The hydrophilicity / hydrophobicity of humanized antibodies was detected by HIC assay. The detection method was as follows: a hydrophobic column (TOSOH Tskgel Buty-NPR(2.5), 4.6) from Tosoh Corporation was used. 100) Hydrophilicity / hydrophobicity was determined using an Agilent HPLC system. Mobile phase A was 1.5 M (NH4)2SO4, and mobile phase B was 25 mM Na2HPO4 (pH=7.0) + 25% IPA. Instrument parameters were set as follows: sample chamber temperature: 8℃, column temperature: 30℃, flow rate: 0.5 mL / min, detection wavelength: 280 nm. The 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.

[0300] 3) The Tm value of the humanized antibody was determined using DSF to reflect the thermostability of the antibody. The experimental steps were as follows: The humanized antibody sample to be tested was diluted to 1 mg / mL with PBS; the dye SYPRO Orange dye (Thermo#56651) was diluted to 40X with ddH2O; the reaction system was: 12.5 uL of sample + 2.5 uL of 40X dye + 5 uL of ddH2O; the membrane was sealed and centrifuged briefly; the sample was detected by Q-PCR. The Q-PCR parameters were set as follows: Target (ROX), program (25℃, 3 min; 1% rate, 95℃; 95℃, 2 min).

[0301] The results of SEC, HIC, and Tm values ​​are shown in Table 5. SEC results indicate that the purity of both humanized antibodies is high, >90%; HIC results indicate that both humanized antibodies bind weakly to the hydrophobic chromatographic column, have short retention times, and exhibit good hydrophilicity; Tm values ​​indicate that both humanized antibodies possess good thermal stability.

[0302] Table 5. Quality Identification of Humanized Antibodies

[0303] 2.6 Cross-detection of anti-c-Met antibody with rat and mouse cMet: Antigen protein Rat-cMet-hFc was diluted 0.5 μg / mL with CBS, coated with the antigen, and blocked with 2% BSA (in PBS) at 37°C for 2 hours. Serially diluted humanized antibody (biotin-labeled) was added (2 μg / mL starting, 3-fold dilution, 11 concentration points), and incubated at 37°C for 2 hours. HRP-labeled anti-biotin secondary antibody (Proteintech, sa00001-0) was added, and incubated at 37°C for 1 hour. TMB substrate was added for color development, and the reaction was stopped with 2M HCl. The absorbance was read at 450 nM. Anti-His-hFc protein (from Chengdu Apak Biotechnology Co., Ltd.) was diluted 0.5 μg / mL with CBS, coated, and blocked with 2% BSA (in PBS) at 37°C for 2 hours. 1 μg / mL mouse cMet-ECD-His... Incubate at 37°C for 2 hours. After 2 hours, add serially diluted humanized antibody (2 ug / mL starting, 3-fold dilution, 11 concentration points). Incubate at 37°C for 2 hours. Add HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164). Incubate at 37°C for 1 hour. Add TMB substrate for color development. Stop with 2M HCl and read the absorbance at 450 nM.

[0304] The experimental results are shown in Figures 1 and 2. The two humanized antibodies, 45A5G10-Hz and 55A10G6-Hz, do not cross-link with rat or mouse cMet (i.e., neither binds to rat or mouse cMet).

[0305] 2.7 Evaluation of anti-c-Met antibody endocytosis activity: MKN45 cells were collected by trypsin digestion and centrifugation. Cells were incubated with the humanized antibody (10 μg / mL) at 4°C for 1 hour, washed three times with PBS, and resuspended in DMEM pre-warmed to 37°C with 10% FBS. The cells were divided into three aliquots and incubated at 37°C for 0, 2, and 4 hours respectively. After pre-cooling and washing three times with PBS, 1.2 μL of anti-human APC fluorescent secondary antibody was added, and the cells were incubated at 4°C for 0.5 hours. After washing three times with PBS, the cells were resuspended and processed. The endocytosis rate was calculated using the following formula: Endocytosis rate (%) = [1 - (Average fluorescence value of the sample detected at this time point - Average fluorescence value of the negative control sample at this time point) / (Average fluorescence value of the sample detected at 0 hours - Average fluorescence value of the negative control sample at 0 hours)] 100.

[0306] The experimental results are shown in Table 6. The endocytosis activity of antibodies 45A5G10-Hz and 55A10G6-Hz was higher than that of ABT700 at the same time, and the endocytosis rate could reach more than 60% after 4 hours.

[0307] Table 6. Detection of endocytic activity of humanized antibodies

[0308] 2.8 Evaluation of anti-c-Met antibody epitope competition: Anti-His-Rabbit Fc protein was coated with CBS at 0.5 μg / ml and blocked with 2% BSA (in PBS) at 37°C for 2 hours. Then, 1 μg / ml h.cMet-ECD-His was added and incubated at 37°C for 2 hours. After 2 hours, serially diluted humanized antibody to be tested (starting at 10 μg / mL, 3-fold dilution, 11 concentration points) was added to achieve a final concentration of 5 μg / mL. The mixture was incubated at room temperature for 0.5 hours. After incubation, 200 ng / ml ABT700-Biotin was added to achieve a final concentration of 100 ng / mL. The mixture was incubated at room temperature for 2 hours. After 2 hours, HRP-labeled anti-biotin secondary antibody (proteintech, sa00001-0) was added and incubated at 37°C for 1 hour. TMB substrate was added for color development, and the reaction was stopped with 2M HCl. The absorbance was then read at 450 nM.

[0309] The experimental results are shown in Figure 3. Neither the humanized antibodies 45A5G10-Hz nor 55A10G6-Hz competed with ABT700 for epitopes.

[0310] 2.9 Evaluation of the competitive effect of anti-c-Met antibody against HGF ligand: cMET is a tyrosine kinase receptor expressed on the cell membrane. It binds to the ligand HGF through the Sema domain, thereby triggering a downstream phosphorylation cascade reaction and ultimately promoting cell proliferation. Therefore, the competitive effect of humanized antibody on ligand binding and its effect on inhibiting HGF-induced ERK phosphorylation were examined.

[0311] MKN45 cells were collected by trypsin digestion and centrifugation, washed three times with pre-chilled PBS, and resuspended in 1% BSA (in PBS) at 2^10 cells per well. 5 50 μL of cells were seeded into 96-well conical plates. Anti-cMet humanized antibody was serially diluted with 1% BSA, starting at 15 μg / ml and ending at 5 μg / ml, with 8 dilution points in a 3-fold dilution gradient. 50 μL of the diluted antibody was mixed with the cells in the conical plate. HGF-His-biotin (from Chengdu Apak Biotechnology Co., Ltd.) was diluted with 1% BSA to 150 ng / ml, and 50 μL of each well was added to the plate to make the final ligand concentration 50 ng / ml. After thorough mixing, the plate was incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 100 μL of 1% BSA (containing 1 μL of antibiotin PE fluorescent secondary antibody, BioLegend, catalog number 405204) was added to each well. The plate was incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry (Beckman, Cytoflex).

[0312] The experimental results are shown in Table 7. Both humanized antibodies exhibited competitive activity against HGF ligand on MKN45 cells, and their competitive activity was higher than that of ABT700.

[0313] Table 7. Detection of competition between humanized antibodies and HGF ligands.

[0314] 2.10 Anti-c-Met antibody inhibits HGF-induced ERK phosphorylation. MDA-MB-468 cells (ATCC, catalog number HTB-132) were digested with trypsin and counted. Cells were resuspended in L15 medium to 2... Cells were cultured at 10^5 / ml and seeded into 24-well plates, 1 ml of cell suspension per well, for a total of 4 wells. The cells were then incubated overnight at 37°C. After 16 hours, the cells from the 6 wells were divided into groups: untreated, HGF (Sinochem Biotech, catalog number: 10463-HNAS), HGF + isotype control (hIgG1, prepared by Suzhou Yilian Biopharmaceutical Co., Ltd.), HGF + 45A5G10-Hz, and HGF + 55A10G6-Hz. The corresponding sterile sample was added to the corresponding well. The test antibody (final concentration 15 ug / ml) was added 15 min beforehand, followed by HGF at a final concentration of 100 ng / ml 15 min later. The cells were then incubated at 37°C for 15 min. After 15 min, the cells were washed three times with PBS, and SDS lysis buffer was added directly to each well to lyse the cells and harvest proteins. The harvested proteins were subjected to routine SDS-PAGE and Western blot analysis. The antibody for detecting phosphorylated ERK was from CST (catalog number #5726S), and the antibody for detecting GAPDH was from Sinocare (catalog number 100242-MM05). Both primary antibodies were diluted 1:2000 and incubated overnight at 4 degrees Celsius.

[0315] As shown in Figure 4, HGF treatment alone can significantly induce ERK phosphorylation in MDA-MB-468 cells, while the addition of antibodies 45A5G10-Hz and 55A10G6-Hz in advance can completely block HGF-induced ERK phosphorylation.

[0316] 2.11 Humanized antibody-induced cMet protein degradation: MKN-45 cells were digested with trypsin and counted. Cells were resuspended in RPMI 1640 + 10% FBS (fetal bovine serum) + Ps (penicillin-streptomycin) to 2... 10^5 / ml of antibody was added, and cells were seeded into 24-well plates, 1 ml of cell suspension per well, for a total of 4 wells. The cells were then placed back into cell culture medium at 37°C and incubated overnight. After 16 hours, the cells from the 4 wells were divided into groups: untreated, isotype control (hIgG1), 45A5G10-Hz, and 55A10G6-Hz. The corresponding sterile sample was then added to the corresponding cell well, with a final antibody concentration of 20 μg / ml. The cells were then placed back into cell culture medium at 37°C and incubated for 60 hours. After 60 hours, the cells were washed three times with PBS, and SDS lysis buffer was added directly to each well to lyse the cells and harvest proteins. The harvested proteins were subjected to routine SDS-PAGE and Western blot analysis. The antibody for detecting cMet was from ProteinTech (catalog number 25869-1-AP), and the antibody for detecting GAPDH was from Sinocare (catalog number 100242-MM05). Both primary antibodies were diluted 1:2000 and incubated overnight at 4 degrees Celsius.

[0317] As shown in Figure 5, compared with the isotype control antibody IgG1 treatment, anti-cMet antibodies 45A5G10-Hz and 55A10G6-Hz can significantly induce cMet degradation.

[0318] Example 3, Preparation of anti-c-Met ADC Example 3.1 Preparation of anti-c-MET-B81 ADC Ab is a c-MET antibody (e.g., 45A5G10-Hz and 55A10G6-Hz as described below), and q can be determined by the data provided below.

[0319] 3.1.1 Preparation of 45A5G10-HZ-B81 (DAR8) Sample: 20 mg of anti-c-Met antibody 45A5G10-HZ was added to a final concentration of 1 mM sodium edetate solution and mixed well. The pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution. 6.5 molar equivalents of the antibody (20 mmol / L) of TCEP solution were added, mixed well, and incubated at 37°C for 90 minutes. 15 molar equivalents of the antibody (10 mmol / L) of B81 dissolved in dimethyl sulfoxide (prepared according to DL-037 in Example 2.37 of WO2022170971) were added to the above solution system, mixed well, and incubated at 37°C for 3 hours to obtain the conjugated sample. After the reaction, the sample was replaced with 20 mM histidine buffer at pH 6.0 using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain an ADC containing anti-c-Met antibody. A solution of 45A5G10-HZ-B81 (DAR8).

[0320] The DAR value of the coupled samples was determined using RP-LC / MS. LC / MS model:

[0321] Liquid phase parameters

[0322] The results showed that the light chain of the 45A5G10-HZ-B81(DAR8) sample was coupled with 0-1 toxin molecules (LC, DAR1 ratios were 0% and 100.0%, respectively), and the heavy chain was coupled with 0-3 toxin molecules (mAb, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100.0%, respectively). Therefore, the coupling ratio (DAR value) of the 45A5G10-HZ-B81(DAR8) sample was calculated to be 8.0. This suggests that q is 8.

[0323] In the preceding text, mAb represents an unconjugated monoclonal antibody; LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate containing one toxin molecule conjugated to either the light or heavy chain; DAR2 represents a conjugate containing two toxin molecules conjugated to either the light or heavy chain; and DAR3 represents a conjugate containing three toxin molecules conjugated to either the light or heavy chain. The theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform. The same applies to mAb, LC, HC, DAR1, DAR2, and DAR3 in the following text.

[0324] 3.1.2 Preparation of 45A5G10-HZ-B81 (DAR4) Samples: 20 mg of anti-c-Met antibody 45A5G10-HZ was added to a final concentration of 1 mM sodium edetate solution and mixed well. The pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution. 20 mmol / L TCEP solution (2.7 molar equivalents of the antibody) was added, mixed well, and incubated at 5°C for 5 h. 6.5 molar equivalents of B81 (10 mmol / L dissolved in dimethyl sulfoxide, refer to WO2022170971) were added to the above solution system. The sample was prepared using DL-037 in Example 2.37. After mixing and standing at room temperature for 1 hour, the coupled sample was obtained. After the reaction was completed, the sample was replaced with a 20mM histidine buffer at pH 6.0 using a 30kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC45A5G10-HZ-B81 (DAR4).

[0325] The DAR value of the coupled sample was determined using Native-LC / MS. LC / MS model:

[0326] Liquid phase parameters

[0327] The results showed that the entire antibody conjugation of the 45A5G10-HZ-B81(DAR4) sample consisted of 0-8 toxin molecules (the proportions of mAb, q=2, q=4, q=6, and q=8 were 0%, 22%, 56%, 23%, and 0%, respectively). Therefore, the conjugation ratio (DAR value) of the 45A5G10-HZ-B81(DAR4) sample was calculated to be 4.0.

[0328] 3.1.3: Preparation of 55A10G6-HZ-B81 (DAR8): 20 mg of anti-c-Met antibody 55A10G6-HZ was added to a final concentration of 1 mM sodium edetate solution and mixed well. The pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution. 6.5 molar equivalents of the antibody (20 mmol / L) of TCEP solution were added, mixed well, and incubated at 37°C for 90 minutes. 15 molar equivalents of the antibody (10 mmol / L) of B81 dissolved in dimethyl sulfoxide were added to the above solution system, mixed well, and incubated at 37°C for 3 hours to obtain the conjugated sample. After the reaction, the sample was replaced with 20 mM histidine buffer at pH 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 ADC55A10G6-HZ-B81 (DAR8).

[0329] Referring to section 3.1.1 above, the DAR value of the coupled samples was determined by LC / MS. For sample 55A10G6-HZ-B81, the light chain was coupled with 0-1 toxin molecules (LC, DAR1 ratios were 0%, 100.0%), and the heavy chain was coupled with 0-3 toxin molecules (mAb, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100.0%). Therefore, the coupling ratio (DAR value) of sample 55A10G6-HZ-B81 was calculated to be 8.0. It can be deduced that q is 8.

[0330] Example 3.2 Preparation of anti-c-MET-vc-MMAE Where Ab is a c-MET antibody (such as ABT700 mentioned below), and q can be determined by the data provided below.

[0331] 3.2.1 Preparation of ABT700-vc-MMAE (DAR9.7) Samples: 20 mg of anti-c-Met antibody ABT700 was added to a final concentration of 1 mM sodium edetate solution and mixed well. The pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution. 6.5 molar equivalents of the antibody (20 mmol / L TCEP solution) were added, mixed well, and incubated at 37°C for 90 minutes. 15 molar equivalents of the antibody (10 mmol / L vc-MMAE dissolved in dimethyl sulfoxide, CAS No.: 646502-53-6, purchased from MedChemExpress) were added to the above solution system, mixed well, and incubated at 37°C for 3 hours to obtain the conjugated sample. After the reaction, the sample was replaced with 20 mM histidine buffer at pH 6.0 using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain an ADC containing anti-c-Met antibody. ABT700-vc-MMAE(DAR9.7) solution.

[0332] Referring to section 3.1.1 above, the DAR value of the coupled samples was determined by LC / MS. The light chain of the ABT700-Vc-MMAE (DAR9.7) sample was coupled with 1-3 toxin molecules (DAR1, DAR2, and DAR3 proportions were 64.3%, 29.8%, and 5.9%, respectively), and the heavy chain was coupled with 3-4 toxin molecules (DAR3 and DAR4 proportions were 56.4% and 43.6%, respectively). Therefore, the coupling ratio (DAR value) of the ABT700-Vc-MMAE (DAR9.7) sample was calculated to be 9.7.

[0333] 3.2.2 Preparation of ABT700-vc-MMAE (DAR4) Sample: 20 mg of anti-c-Met antibody ABT700 was added to a final concentration of 1 mM sodium edetate solution and mixed well. The pH of the sample was adjusted to 7.5 with 0.5 M disodium hydrogen phosphate solution. 20 mmol / L TCEP solution (2.8 molar equivalents of the antibody) was added, mixed well, and incubated at room temperature for 90 minutes. 4.2 molar equivalents of the antibody (10 mmol / L vc-MMAE dissolved in dimethyl sulfoxide, CAS No.: 646502-53-6, purchased from MedChemExpress) were added to the above solution system, mixed well, and incubated at room temperature for 3 hours to obtain the conjugated sample. After the reaction, the sample was replaced with 20 mM histidine buffer at pH 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).

[0334] Referring to section 3.1.1 above, the DAR value of the coupled samples was determined by LC / MS. The light chain of the ABT700-VC-MMAE(DAR4) sample was coupled with 0-1 toxin molecules (LC, DAR1 ratio was 52.2%, 47.8%, respectively), and the heavy chain was coupled with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 ratio was 17.5%, 47.2%, 22.9%, 12.5%, respectively). Therefore, the coupling ratio (DAR value) of the ABT700-VC-MMAE(DAR4) sample was calculated to be 4.0.

[0335] Example 4. In vitro evaluation of anti-human c-Met antibody and its ADC 4.1 Detection of protein affinity of anti-human c-Met ADC Protein: 0.5 μg / ml of anti-His-RabbitFc protein coated with CBS was added, and the mixture was blocked with 2% BSA (in PBS) at 37°C for 2 hours. Then, 1 μg / ml of h.cMet-ECD-His was added and incubated at 37°C for 2 hours. After 2 hours, serially diluted antibody or ADC to be tested (2 μg / mL starting, 3-fold dilution, 11 concentration points) was added and incubated at 37°C for 2 hours. After 2 hours, HRP-labeled anti-human specific secondary antibody (Jackson, 115-035-164) was added and incubated at 37°C for 1 hour. TMB substrate was added for color development, and the reaction was stopped with 2M HCl. The absorbance was then read at 450 nM.

[0336] The experimental results are shown in Table 8. The antigen binding affinity of 45A5G10-HZ, 45A5G10-HZ-B81(DAR8), 55A10G6-Hz, and 55A10G6-HZ-B81(DAR8) did not change significantly, while the antigen binding affinity decreased significantly after ABT700 was coupled into an ADC.

[0337] Table 8. Protein affinity assays before and after cMET antibody conjugation into ADC.

[0338] 4.2 Anti-human cMet ADC Cell Affinity Assay 1. Trypsin Digestion and Centrifugation: Collect NCI-H358 (human non-small cell lung cancer cells, purchased from ATCC, catalog number CRL-5807), LS1034 (human colon adenocarcinoma cells, Nanjing Kebai, catalog number CBP60013), NCI-H69 (human small cell lung cancer cells, Wuhan Pronosai, catalog number CL-0677), and NCI-H716 (human colorectal adenocarcinoma cells, purchased from ATCC, catalog number CCL-251) cells directly by centrifugation, wash three times with pre-cooled PBS, resuspend the cells in 1% BSA (in PBS), and divide into 2 cells per well. 10 550 μL of cells were seeded into 96-well conical plates. The anti-cMet humanized antibody ADC was serially diluted with 1% BSA, starting at 15 μg / ml, with 4-fold dilutions and 8 dilution points. 50 μL of the diluted ADC was mixed with the cells in the conical plate and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 100 μL of 1% BSA (containing 1 μL of anti-human APC fluorescent secondary antibody, BioLegend, catalog number 410712) was added to each well and incubated at 4°C for 0.5 hours. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry (Beckman, Cytoflex).

[0339] The experimental results are shown in Table 9. The two humanized antibodies, conjugated with B81, showed high affinity for NCI-H358, LS1034, NCI-H69, and NCI-H716 cells. Furthermore, the affinity of the two antibody ADCs for all three tumor cell types was higher than that for ABT700-VcMMAE (DAR9.7). The ADCs of both antibodies showed higher affinity for NCI-H716 cells than ABT700-VcMMAE (DAR4).

[0340] Table 9. Cell affinity assay for anti-human cMet ADC

[0341] Test 2: Trypsin digestion and centrifugation were used to collect MKN45 cells (derived from JCBR JCRB0254). The cells were washed three times with pre-cooled PBS and diluted to 1×10⁻⁶. 6 Cells / mL, 1×10⁶ cells ...well 5 100 μL of cells were seeded into 96-well conical plates. 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ were serially diluted with FACS buffer, starting at 25 nM, with 4-fold dilutions and 8 dilution points. 100 μL of each cell was added to the conical plate and mixed with the cells. The cells were incubated at 4°C for 0.5 h. After washing three times with pre-chilled PBS, 100 μL of fluorescent secondary antibody PE anti-human IgG diluted with ice-cold FACS buffer was added to each well. The cells were incubated at 4°C for 0.5 h. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry.

[0342] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ exhibited high binding activity to MKN45, and their affinities for MKN45 were comparable. 50 The values ​​are 0.215 nM and 0.189 nM, respectively.

[0343] 4.3 In vitro killing activity assay of ADC: 1. Collect tumor cells in good growth condition by digestion and centrifugation. SW480 (purchased from ATCC, catalog number CCL-228), NCI-H358 (purchased from ATCC, catalog number CRL-5807), LS1034 (Nanjing Kebai, catalog number CBP60013), and NCI-H716 (purchased from ATCC, catalog number CCL-251) cells were directly collected by centrifugation. All cells were resuspended in RPMI 1640 + 5% FBS + Ps, counted, and seeded at 5000 (LS1034 and NCI-H716) or 3000 (SW480 and NCI-H358) cells per well, with a volume of 100 uL. The ADC to be tested was diluted with the resuspension medium corresponding to the above three cell types, starting at 300 μg / mL (2 μM), 3-fold dilution, 11 concentration points, and 100 μL of each was taken. Add μL / well to the plate to bring the final ADC concentration to 150 μg / mL. Incubate at 37°C for 5 days (LS1034, NCI-H358) or 7 days (NCI-H716, SW480). After incubation, add 20 μL / well of CCK8 and react for 1-3 hours. Read the values ​​at 450nm using a microplate reader and import the data into Graphpad Prism for curve fitting.

[0344] The experimental results are shown in Table 10. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) effectively killed four types of tumor cells: LS1034, NCI-H358, NCI-H716, and SW480. In LS1034 cells, the cytotoxic activity of 45A5G10-Hz-B81 (DAR8) was comparable to that of ABT700-VcMMAE (DAR9.7), while 55A10G6-Hz-B81 (DAR8) was slightly weaker than ABT700-VcMMAE (DAR9.7). In NCI-H358 cells, 45A5G10-Hz-B81 (DAR8) showed stronger cytotoxic activity than 55A10G6-Hz-B81 (DAR8). In NCI-H716 cells… On 6 cells, 45A5G10-Hz-B81(DAR8) and 55A10G6-Hz-B81(DAR8) showed stronger cytotoxic activity than ABT700-VcMMAE(DAR4); on SW480 cells, 45A5G10-Hz-B81(DAR8) showed stronger cytotoxic activity than ABT700-VcMMAE(DAR4), while 55A10G6-Hz-B81(DAR8) was comparable to ABT700-VcMMAE(DAR4).

[0345] Table 10. Detection of in vitro cell-killing activity of anti-human cMet ADC

[0346] Test 2: NCI-H358 cells (derived from ECACC-95111733), NCI-H441 cells (derived from ATCC-HTB-174), and MKN45 cells (derived from JCBR JCRB0254) were collected by trypsin digestion and centrifugation. Cells were resuspended in RPMI 1640 medium containing 10% FBS, counted, and seeded at 1500 cells per well (NCI-H441 / NCI-H358 / MKN45), with a volume of 135 μL. The test ADC (45A5G10-HZ-B81) starting at 20000 nM was diluted with the corresponding resuspension medium for each of the three cell types, starting with 4-fold dilutions, resulting in 9 concentration points. Further dilution to 500 nM was performed, and 15 μL / well was added to the plate to achieve a final ADC concentration of 50 nM. The plate was incubated at 37°C for 6 days. After incubation, CellTiter-Glo working solution was added, and the plate was incubated at 75°C. μL / well, shake on a track shaker for 2 minutes to induce cell lysis, and incubate at room temperature for 10 minutes to stabilize the luminescence signal; detect the luminescence signal with an enzyme-linked immunosorbent assay (ELISA) reader.

[0347] The experimental results are shown in Table 11. The results indicate that 45A5G10-HZ-B81 (DAR8) exhibits strong anti-proliferative activity in NCI-H441 cells, with an IC50 of 0.031 nM. 45A5G10-HZ-B81 (DAR8) also shows anti-proliferative activity in NCI-H358 cells, with an IC50 of 0.161 nM. Furthermore, 45A5G10-HZ-B81 (DAR8) demonstrates strong anti-proliferative activity in MKN45 cells, with an IC50 of 0.057 nM.

[0348] Table 11. Antiproliferative effects of anti-human cMet ADC

[0349] The results showed that 45A5G10-HZ-B81 (DAR8) significantly inhibited the proliferation of NCI-H441, NCI-H358, and MKN45 cell lines, with maximum inhibition rates of 76.44%, 43.07%, and 88.29%, respectively. The target expression level in NCI-H358 cells was significantly lower than that in NCI-H441 cells (FACS verification of c-Met target expression in cells is shown in Figure 12), yet it still exhibited a maximum inhibition rate of >40%.

[0350] 4.4 Forte bio assays of the affinity of anti-c-Met antibodies and their ADCs for human Fc receptors and complement C1q. 4.4.1: Affinity assays of FcγRI with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. The Fc receptor, FcγRI (CD64), can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to the Fc receptor affects the safety and efficacy of the antibody.

[0351] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRI were detected using a Fortebio Octet molecular interaction analyzer to evaluate their potential ADCC and ADCP activities.

[0352] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRI using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 5 μg / mL solution of FcγRI (purchased from ACRO Biosystems, catalog number FCA-H52H1) was added to the HIS1K sensor to immobilize FcγRI on the sensor surface. The binding and dissociation parameters of the antibody with FcγRI were measured in buffer at antibody concentrations of 200, 100, 50, 25, 12.5, and 6.25 nM. After the sensor immobilized with antigen was equilibrated in buffer for 60 s, the binding time of FcγRI immobilized on the sensor with each antibody was measured at 60 s; the dissociation time of FcγRI from the antibody was measured at 120 s. Data were analyzed using DataAnalysis 11 to obtain the affinity constants of each antibody with FcγRI.

[0353] The results of the affinity constant determination of FcγRI with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 12 below.

[0354] Table 12. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcγRI

[0355] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRI.

[0356] 4.4.2: Affinity detection of FcγRIIIa_V176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Fc receptor FcγRIIIa_V176, also known as (CD16a_V176), can bind to the Fc terminus of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0357] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIIa_V176 were determined using a Fortebio Octet molecular interaction analyzer to evaluate their potential ADCC activity.

[0358] The experimental method for detecting the affinity constant of the corresponding antibody with FcγRIIIa_V176 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 10 μg / mL solution of FcγRIIIa_V176 (purchased from ACRO Biosystems, catalog number CD8-H52H4) was added to the HIS1K sensor to immobilize FcγRIIIa_V176 on the sensor surface. The binding and dissociation parameters of the antibody with FcγRIIIa_V176 were measured in buffer at antibody concentrations of 2500, 1250, 625, 312.5, 156.25, and 78.125 nM. After the sensor immobilized with antigen was equilibrated in buffer for 60 s, the binding time of FcγRIIIa_V176 immobilized on the sensor to each antibody was measured at 60 s; the dissociation time of FcγRIIIa_V176 from the antibody was also measured at 60 s. Data were analyzed using DataAnalysis11 to obtain the affinity constants between each antibody and FcγRIIIa_V176.

[0359] The results of the affinity constant determination of FcγRIIIa_V176 with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 13 below.

[0360] Table 13. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcγRIIIa_V176

[0361] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIIa_V176.

[0362] 4.4.3: Affinity detection of FcγRIIIa_F176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Fc receptor FcγRIIIa_F176, also known as (CD16a_F176), can bind to the Fc terminus of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0363] The binding experiments of the two samples with FcγRIIIa_F176 (purchased from ACRO Biosystems, catalog number CDA-H 522 0) were identical to those of CD16a (V 176) except that the analyte concentration range in the binding step was optimized to (5000, 2500, 1250, 625, 312.5, 156.25 nM).

[0364] The affinity constants of FcγRIIIa_F176 with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 14 below.

[0365] Table 14: Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcγRIIIa_F176

[0366] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIIa_F176.

[0367] 4.4.4: Affinity detection of FcγRIIa_H167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Fc receptor FcγRIIa_H167, also known as (CD32a_H167), can bind to the Fc terminus of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0368] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIa_H167 were determined using a Fortebio Octet molecular interaction analyzer to evaluate their potential ADCC activity.

[0369] The experimental method for detecting the affinity constant of the corresponding antibody with FcγRIIa_H167 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 10 μg / mL solution of FcγRIIa_H167 (purchased from ACRO Biosystems, catalog number CD1H5223) was added to the HIS1K sensor to immobilize FcγRIIa_H167 on the sensor surface. The binding and dissociation parameters of the antibody with FcγRIIa_H167 were measured in buffer at antibody concentrations of 5000, 2500, 1250, 625, 312.5, and 156.25 nM. After the sensor immobilized with antigen was equilibrated in buffer for 60 s, the binding time of FcγRIIa_H167 immobilized on the sensor to each antibody was measured at 60 s; the dissociation time of FcγRIIa_H167 from the antibody was also measured at 60 s. Data were analyzed using DataAnalysis11 to obtain the affinity constants between each antibody and FcγRIIa_H167.

[0370] The results of affinity constant determination for FcγRIIa_H167 with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 15 below.

[0371] Table 15. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcγRIIa_H167

[0372] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIa_H167.

[0373] 4.4.5: Affinity detection of FcγRIIa_R167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Fc receptor FcγRIIa_R167, also known as (CD32a_R167), can bind to the Fc terminus of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0374] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRIIa_R167 were determined using the Fortebio Octet molecular interaction analyzer to evaluate their potential ADCC activity.

[0375] The binding experiments of the two samples with FcγRIIa_R167 (purchased from ACRO Biosystems, catalog number CDA-H 522 1) were conducted with the same binding procedure as those with FcγRIIa_H167.

[0376] The results of the affinity constant determination of FcγRIIa_R167 with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 16 below.

[0377] Table 16. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcγRIIa_R167

[0378] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIa_R167.

[0379] 4.4.6: Affinity detection of FcγRIIb / c with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Fc receptor FcγRIIb / c, also known as (CD32b / c), can bind to the Fc terminus of IgG antibodies, negatively regulating the function of immune cells, inhibiting the activation and proliferation of immune cells, and inhibiting the secretion of cytokines.

[0380] In this experiment, the affinity constants of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ with FcγRIIb / c were determined using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ with FcγRIIb / c.

[0381] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRIIb / c using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 10 μg / mL solution of FcγRIIb / c (purchased from ACRO Biosystems, catalog number CDB-H5228) was added to the HIS1K sensor to immobilize FcγRIIb / c on the sensor surface. The binding and dissociation parameters of the antibodies with FcγRIIb / c were measured in buffer at antibody concentrations of 10000, 5000, 2500, 1250, 625, and 312.5 nM. After the sensor immobilized with antigen was equilibrated in buffer for 60 s, the binding time of FcγRIIb / c immobilized on the sensor to each antibody was measured at 60 s; the dissociation time of FcγRIIb / c from the antibody was also measured at 60 s. The data were analyzed using DataAnalysis11 to obtain the affinity constants of each antibody and FcγRIIb / c.

[0382] The results of the affinity constant determination of FcγRIIb / c with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 17 below.

[0383] Table 17. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcγRIIb / c

[0384] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities with FcγRIIb / c.

[0385] 4.4.7: Affinity detection of FcRn with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Fc receptor FcRn can bind to the Fc terminus of IgG antibody, protecting the antibody macromolecule from destruction, and then release the antibody macromolecule in a blood environment with pH 7.4.

[0386] In this experiment, the affinity constants of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ with FcRn were detected using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ with FcRn.

[0387] The experimental method for detecting the affinity constants of corresponding antibodies and FcRn using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 2 μg / mL FcRn solution (purchased from ACRO) was added to the HIS1K sensor to immobilize the FcRn on the sensor surface. The binding and dissociation parameters of the antibody and FcRn were measured in buffer at antibody concentrations of 1000, 500, 250, 125, 62.5, and 31.25 nM. After the sensor immobilized with antigen was equilibrated in buffer for 60 s, the binding time of the FcRn immobilized on the sensor to each antibody was measured at 60 s; the dissociation time of FcRn from the antibody was also measured at 60 s. Data were analyzed using DataAnalysis 11 to obtain the affinity constants of each antibody and FcRn.

[0388] The results of the affinity constant determination of FcRn with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 18 below.

[0389] Table 18. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with FcRn.

[0390] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities with FcRn.

[0391] 4.4.8: Affinity assay of C1q with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. Serum complement C1q can bind to the Fc terminus of IgG antibodies, mediating the CDC effect. The ability of therapeutic monoclonal antibodies to bind to C1q affects the safety and efficacy of the antibody.

[0392] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with C1q were detected using a Fortebio Octet molecular interaction analyzer to evaluate the CDC activity of each antibody.

[0393] The experimental method for detecting the affinity constant of the corresponding antibody with C1q using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer is a PBS solution containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). 50 μg / mL of antibody is immobilized on the FAB2G sensor at a height of approximately 3.0 nm. The sensor is equilibrated in the buffer for 60 s. The antibody immobilized on the sensor binds to antigen C1q (purchased from Sigma, catalog number C1740-1MG). The antigen concentrations for 45A5G10-HZ are 20, 10, 5, 2.5, 1.25, and 0.625 nM, and the antigen concentrations for 45A5G10-HZ-B81 (DAR8) are 500, 250, 125, 62.5, 31.25, and 15.625 nM, respectively, for 60 s. The antigen and antibody dissociate in the buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 seconds, repeated three times. Data were analyzed using DataAnalysis11 to obtain the affinity constant.

[0394] The results of the affinity constant determination of C1q with 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ are shown in Table 19 below.

[0395] Table 19. Kinetic parameters of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ combined with C1q.

[0396] The results showed that 45A5G10-HZ and 45A5G10-HZ-B81 (DAR8) could bind to C1q, with affinity constants of 3.99E-09M and 2.00E-07M, respectively. The results indicated that the binding activity of 45A5G10-HZ toxin was effectively eliminated after conjugation.

[0397] 4.5 Forte bio assay for ADCC activity of anti-c-Met antibody and its ADC ADCC effect refers to the direct killing of target cells by effector immune cells with cytotoxic activity through the recognition of the Fc fragment of the antibody bound to the target cell antigen by the Fc receptor (FcR) expressed on their surface.

[0398] The ADCC activity of MKN45 cells (derived from Sichuan Sibowo) expressing c-MET antigen was detected using 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ. The specific method is as follows: 0.5 g of BSA was weighed and added to 50 mL of RPMI 1640 basal medium. After thorough dissolution, the solution was filtered through a 0.22 μM microfiltration filter to obtain the sample dilution (RPMI 1640 + 1% BSA), which was prepared fresh for use. The day before the experiment, target cells (MKN45) in the exponential growth phase were digested with 0.25% trypsin in a biosafety cabinet to prepare a single-cell suspension. Cells were counted using a cell counter, and the cell density was adjusted to 2 × 10⁶ cells / cells using RPMI 1640 + 10% FBS medium. 5 Cells / mL, 100 μL / well, were added to cell culture plates and incubated overnight at 37°C in a CO2 cell culture incubator. The supernatant was then discarded. Effector cells (Jurkat-NFAT-CD16a, sourced from Wuhan Taituozhong Biotechnology) in the exponential growth phase were collected and counted using a cell counter. The cell density was adjusted to 4 × 10⁶ cells / well using RPMI 1640 basal medium. 6 50 μL / well was added to wells containing MKN45 cells, i.e., the sample group (ADC and antibody) and the negative control group (hIgG1). Daudi cells (CD20 target cells, derived from ATCC catalog number CCL213) and effector cells (Jurkat-NFAT-CD16a) in the exponential growth phase were collected and counted using a cell counter. Appropriate amounts of Daudi cell and Jurkat-NFAT-CD16a cell suspensions were mixed and the cell density was adjusted with RPMI 1640 basal medium to a final density of 4 × 10⁻⁶ Daudi cells. 5 The final density of Jurkat-NFAT-CD16a cells was 4 × 10⁶ / mL. 6 50 μL / well was added to the positive control wells of the experimental plate. Each test substance was diluted to 2× concentration (40000 ng / mL) with sample diluent, and then further serially diluted 5-fold, for a total of 8 concentrations. Rituximab was serially diluted 7-fold, for a total of 8 concentrations. According to the experimental plate layout, 50 μL of each serially diluted 2× test substance diluent was added to each well. The cell culture plate was placed on a microplate shaker and mixed at 500 rpm for 5 minutes. The 96-well plate was then incubated at 37°C in a CO2 incubator for approximately 6 hours. After incubation, 50 μL / well of pre-melted and equilibrated Bio-Glory one-step reagent (from Adamas Life, catalog number RA-GL04) was added to each well of the cell culture plate, and the mixture was shaken at room temperature for 10 minutes. The biofluorescence signal value was measured using a microplate reader.

[0399] Figure 17 shows the ADCC activity detection results of MKN45 cells expressing c-MET antigen by 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ.

[0400] The results showed that, in the MKN45 and JurkatNFAT-CD16a co-culture system, at the same dose level, the ADCC activity induced by 45A5G10-HZ-B81(DAR8) was significantly lower than that 45A5G10-HZ.

[0401] The results show that 45A5G10-HZ has a weak ADCC effect, while 45A5G10-HZ-B81(DAR8) does not show a significant ADCC effect.

[0402] 4.6 Forte bio assay for CDC activity of anti-c-Met antibodies and their ADCs. The CDC effect is achieved by the antibody binding to the corresponding antigen on the cell membrane surface and simultaneously binding to complement C1q, activating the classical complement-dependent cytotoxic pathway, and then forming a membrane attack complex, thereby exerting a lytic effect on the target cell.

[0403] The CDC activity of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ on MKN45 cells expressing c-MET antigen was detected using the following method: 0.5 g of BSA was weighed and added to 50 mL of RPMI 1640 basal medium. After thorough dissolution, the solution was filtered through a 0.22 μM microfilter to obtain a sample dilution (RPMI 1640 + 1% BSA), which was prepared fresh for use. Target cells (MKN45 and Daudi) in the exponential growth phase were collected, prepared into single-cell suspensions, and counted using a cell counter. The MKN45 cell population was adjusted to 2 × 10⁶ cells / cells using RPMI 1640 basal medium. 5 Daudi cells were adjusted to a density of 6 × 10⁶ cells / mL using RPMI 1640 basal medium. 5Cells / mL were mixed thoroughly by pipetting, and 40 µL of cell suspension was added to each well of the cell culture plate according to the experimental plate layout. One bottle of lyophilized guinea pig serum complement (from BERSEE catalog number BM361Y) was taken and reconstituted with 1 mL of DMEM basal medium, at which point the complement concentration was 100%. 1000 µL of 100% complement was added to 1000 µL of DMEM basal medium and mixed thoroughly, at which point the complement concentration was 50%. 20 µL of 50% serum complement was added to each well of the cell culture plate to bring the final complement concentration in the well to 10%. Each test sample was diluted to a concentration of 2.5× (i.e., 250,000 ng / mL) with sample diluent, and then further serially diluted 3-fold to a total of 12 concentrations. 40 µL of each test sample diluent was added to each well. The cell culture plate was placed on a microplate shaker and mixed at 500 rpm for 5 minutes, then incubated at 37°C in a CO2 incubator for approximately 6 hours. After incubation, 50 µL of pre-melted and equilibrated Cell Titer Turbo 2.0 reagent (at room temperature) was added to each well of the cell culture plate, and the mixture was shaken at room temperature for 10 minutes. The biofluorescence signal value was then measured using a microplate reader.

[0404] Figure 18 shows the results of CDC activity assays using 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ on MKN45 cells expressing c-MET antigen. The results indicate that, in the presence of complement, neither 45A5G10-HZ-B81 (DAR8) nor 45A5G10-HZ mediated CDC effects at any dose level.

[0405] 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), and mouse c-MET (purchased from Sino Biological, catalog number 50622-M08H) were diluted with CBS at 1 μg / mL, then coated with the antigen, and blocked with 2% BSA (in PBS) at 37°C for 1.5 hours. Serially diluted 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ (starting at 1 μg / mL, 4-fold dilution, 8 concentration points) were added, and incubated at 37°C for 1 hour. HRP-labeled secondary antibody (Goat Anti-Human) was then added. IgG and Monkey ads-HRP (purchased from Southern Biotech, catalog number F4522-V172E) were incubated at 37°C for 1 hour, followed by the addition of TMB substrate for color development. After stopping the reaction with stop solution, the OD values ​​of each well were read at a wavelength of 450 nm (reference wavelength 630 nm). The experimental results are shown in Table 20.

[0406] Table 20. Cross-reaction of species 45A5G10-HZ or 45A5G10-HZ-B81 (DAR8)

[0407] 45A5G10-HZ or 45A5G10-HZ-B81 (DAR8) specifically binds to human c-MET and monkey c-MET only. The EC50 values ​​of 45A5G10-HZ binding to human c-MET and monkey c-MET species proteins are 25.43 ng / mL and 29.64 ng / mL, respectively. The EC50 values ​​of 45A5G10-HZ-B81 (DAR8) binding to human c-MET and monkey c-MET species proteins are 23.63 ng / mL and 32.42 ng / mL, respectively.

[0408] The results showed that the binding ability of 45A5G10-HZ-B81 (DAR8) to c-MET antigens of different species was similar to that of 45A5G10-HZ.

[0409] Example 5, In vivo evaluation of anti-human cMet-ADC 5.1 Efficacy test of anti-human cMet-ADC against non-small cell lung cancer xenografts Test 1 To verify the in vivo efficacy of anti-human cMet ADC, the antitumor effect of the test drug in subcutaneous xenograft female Balb / cNude mouse models was evaluated. 5-6 week old female Balb / c Nude mice (Vitallix) were purchased. Human non-small cell lung cancer NCI-H358 cells, which had reached the logarithmic growth phase, were digested with EDTA and then resuspended in PBS. Each mouse was subcutaneously inoculated with 5 10 6 Cells, waiting for the tumor to grow to 100-200m 3 The dosage is 1 mg / kg or 3 mg / kg, administered intravenously once a week.

[0410] The main observation indicators of this experiment are: 1) TGI (%), calculated by the formula: TGI(%)=(1-T / C)×100% (T and C are the relative tumor volumes of the treatment group and the control group at a specific time point, respectively); 2) Photographs of tumor volume and weight at the end of the experiment.

[0411] The experimental results are shown in Table 21 and Figure 6. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) showed good tumor-suppressing effects in mice, and at a dose of 1 mg / kg, they were more effective than ABT700-VcMMAE. The changes in mouse body weight are shown in Figure 7, indicating that the ADC had no effect on mouse body weight throughout the administration process.

[0412] Table 21. In vivo efficacy of anti-human cMet ADC in NCI-H358 CDX model

[0413] The purpose of this experiment was to evaluate the antitumor efficacy of test sample 45A5G10-HZ-B81 (DAR8) in a human non-small cell lung cancer cell NCI-H358 xenograft model.

[0414] Female BALB / c Nu nude mice were subcutaneously inoculated with NCI-H358 cells, and tumors were allowed to grow to 180 mm. 3 Left and right (not exceeding 200 mm) 3Fifty-six animals were selected and divided into seven groups: a Vehicle group (saline), a small molecule toxin released by 45A5G10-HZ-B81 (DAR8), prepared according to Example A1.9 of WO2022170971, at a dose of 0.07 mg / kg; an IgG1-B81 (isotype control antibody ADC, prepared according to IgG1-ADC-07 in Example 4.6.1 of WO2022170971) at a dose of 3 mg / kg; and 45A5G10-HZ-B81 (DAR8) at doses of 0.3, 1, and 3 mg / kg, with eight animals in each group. The drugs were administered via tail vein injection once weekly (QW) for three consecutive weeks. The day of grouping was designated as Day 1 of the experiment. Drug administration began on Day 1 and ended on Day 22 (22 days after the first administration). Animals were euthanized, tumors were isolated, and weighed.

[0415] The results are shown in Table 22 and Figure 13. The tumor volume in the 0.3, 1, and 3 mg / kg dose groups of 45A5G10-HZ-B81 (DAR8) was significantly lower than that in the Vehicle group (P<0.01 or P<0.001), with tumor inhibition rates (TGI) of 43.4%, 68.0%, and 81.1%, respectively, showing a positive correlation with the dose. The tumor volume in the 3 mg / kg IgG1-B81 group was significantly lower than that in the Vehicle group (P<0.01), with a TGI of 49.3%. These results indicate that the anti-c-Met antibody drug conjugate and its drug linker (toxin-linker) disclosed herein can achieve anti-tumor effects without requiring antibody endocytosis or positive antigen expression in tumor cells.

[0416] The animal weight measurement results are shown in Figure 14. At the end of the experiment (Day 22), the average weight and weight change rate of animals in each dose group of 45A5G10-HZ-B81 (DAR8) continued to increase during the administration period, and there was no significant difference compared with the Vehicle group (P>0.05).

[0417] Table 22 In vivo efficacy of anti-human cMet ADC in NCI-H358 xenograft model

[0418] 5.2 Efficacy testing of anti-human cMet-ADC in human colon cancer CDX model: The anti-tumor effect of anti-cMet-ADC in female BALB / c nude mouse animal model of subcutaneous xenograft of human colon cancer SW480 cell line was evaluated using a similar method.

[0419] The experimental results are shown in Table 23 and Figure 8. 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) both showed good tumor-suppressing effects on tumor cells with low human cMet expression in mice. At a dose of 5 mg / kg, the TGI was significantly higher than that of ABT700-VcMMAE (DAR4). The changes in mouse body weight are shown in Figure 9, indicating that the ADC had no effect on mouse body weight throughout the entire administration process.

[0420] Table 23. In vivo efficacy of anti-human cMet ADC in SW480 CDX model

[0421] 5.3 Efficacy testing of anti-human cMet-ADC in human colorectal adenocarcinoma CDX model: The antitumor effect of the cMet-ADC test drug in the subcutaneous xenograft of human colorectal adenocarcinoma NCI-H716 cell line in female BALB / c nude mouse animal model was evaluated using a similar method.

[0422] The experimental results are shown in Table 24 and Figure 10. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) showed good tumor-suppressing effects in mice, and their TGI was significantly higher than that of ABT700-VcMMAE (DAR4) at a dose of 1 mg / kg. The changes in mouse body weight are shown in Figure 11, indicating that the ADC had no effect on mouse body weight throughout the entire administration process.

[0423] Table 24. In vivo efficacy of anti-human cMet ADC in NCI-H716 CDX model

[0424] 5.4 Efficacy test of anti-human cMet-ADC in human colorectal adenocarcinoma PDX model The purpose of this experiment was to evaluate the antitumor effect of the test product 45A5G10-HZ-B81 (DAR8) in the human colorectal cancer CR5088 PDX model (derived from Crown Bioscience Inc.).

[0425] NOD / SCID mice were subcutaneously inoculated with CR5088 tumor blocks (derived from Crown Bioscience, Inc.) to establish a human subcutaneous xenograft model of colorectal cancer. The experiment consisted of four groups (n=8 per group) with three dosage groups of the test drug 45A5G10-HZ-B81 (DAR8) (1 mg / kg, 3 mg / kg, and 10 mg / kg) and a solvent control group. Administration was once weekly for three weeks. Efficacy was evaluated based on tumor inhibition rate (TGI) calculated from tumor volume, and safety was evaluated based on changes in animal weight and mortality.

[0426] The tumor volume results are shown in Table 25 and Figure 15. 45A5G10-HZ-B81 (DAR8) showed a trend of inhibiting tumor growth at doses of 1 mg / kg, 3 mg / kg and 10 mg / kg, with tumor inhibition rates of 41.54%, 95.89% and 97.56%, respectively. The 3 mg / kg and 10 mg / kg dose groups showed statistically significant differences compared with the blank control group, with P values ​​less than 0.001.

[0427] The results of the animal weight changes are shown in Figure 16. No mice in any group experienced severe weight loss (BWL < 15%), and no mice died unexpectedly during the experiment, indicating good tolerance.

[0428] Table 25. Efficacy of anti-human cMet-ADC in human colorectal adenocarcinoma PDX model

[0429] 5.5 Efficacy test of anti-human cMet-ADC against gastric cancer xenografts The purpose of this experiment was to evaluate the antitumor efficacy of the test product 45A5G10-HZ-B81 (DAR8) in a human gastric cancer MKN45 xenograft model.

[0430] Female NCG mice (Beijing Chuangmo) were subcutaneously inoculated with MKN45 cells (derived from JCBR JCRB0254) to establish a human gastric cancer model. The experiment was divided into four groups: a saline control group, a toxin treatment group (45A5G10-HZ-B81(DAR8) released toxin, 0.23 mg / kg), a 45A5G10-HZ (10 mg / kg) and IgG1-B81 (10 mg / kg) treatment group, a 45A5G10-HZ-B81(DAR8) (1 mg / kg) treatment group, a 45A5G10-HZ-B81(DAR8) (3 mg / kg) treatment group, and a 45A5G10-HZ-B81(DAR8) (10 mg / kg) treatment group, with eight mice in each group. Efficacy was evaluated based on the tumor growth index (TGI), and safety was evaluated based on changes in animal body weight and mortality.

[0431] The tumor volume results are shown in Table 26 and Figure 19. 45A5G10-HZ showed an inhibitory effect on the growth of human gastric cancer MKN45 subcutaneous xenografts. 45A5G10-HZ-B81 (DAR8) administered via tail vein injection at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, once weekly for three consecutive weeks, significantly inhibited the growth of human gastric cancer MKN45 subcutaneous xenografts, with relative tumor inhibition rates (TGI) of 72%, 96%, and 98%, respectively, all statistically significant compared to the solvent control group. The tumor volume in the IgG1-B81 10 mg / kg group was significantly lower than that in the physiological group (P<0.01), with a tumor inhibition rate (TGI) of 75%. This indicates that the anti-c-Met antibody-drug conjugate and its drug linker (toxin-linker) disclosed herein can achieve anti-tumor effects without requiring antibody endocytosis or positive antigen expression on tumor cells.

[0432] Table 26: Efficacy test data of NCI-H358 cell xenograft model

[0433] Note: 1. Data are expressed as mean ± standard error.

[0434] 2. T / C % = TRTV / CRTV × 100%; TGI% = (1-T / C) × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the solvent control group; RTV=Vt / V0, where V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment).

[0435] Example 6. Plasma stability test of C-MET antibody-drug conjugate: The stability of 45A5G10-HZ-B81(DAR8) in plasma was evaluated by measuring the release of bioactive molecular toxins in human plasma incubated with 45A5G10-HZ-B81(DAR8).

[0436] Experimental system information

[0437] 1. Experimental Procedure: Plasma Preparation: Thaw the frozen plasma rapidly at 37°C. Plasma should be placed on ice before use.

[0438] Thawed plasma was filtered through a 0.22 μm filter membrane and used immediately after filtration.

[0439] Drug dilution process: Step 1: Prepare 1 mg / mL drug diluent: Take a certain volume of 26.6 mg / mL 45A5G10-HZ-B81 (DAR8) and add it to the corresponding volume of 0.1M PBS to prepare a 1 mg / mL drug diluent. Filter it through a 0.22 μm filter membrane before use.

[0440] Step 2: Prepare stability samples: Take a certain volume of plasma or 0.1M PBS and add it to the drug diluent to prepare a system with a concentration of 100 μg / mL. Mix gently, aliquot 200 µL into EP tubes (centrifuge tubes) (this operation is performed on ice at all time points), and seal them.

[0441] 0 min sample: After the corresponding sample is aliquoted in step 2, add 5 times the volume of methanol immediately, vortex mix for 2 min, centrifuge (4℃, 17000×g) for 10 min, and take the supernatant. Store the supernatant at -60℃ or below for testing.

[0442] The prepared stability samples were incubated at 37°C. After incubation at 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, the samples were precipitated with 5 times their volume of methanol. The supernatant was then frozen at -60°C for analysis. Free small molecules were detected by LC-MS / MS.

[0443] 2. Sample detection: The concentration of small molecule drugs in all samples of each group was determined by LC-MS / MS.

[0444] 3. Data Processing: Calculation of Theoretical Total Concentration of Free Small Molecule: Ctotal (toxin) = (Cdose concentration (45A5G10-HZ-B81(DAR8)) / molecular weight 45A5G10-HZ-B81(DAR8)) × antibody-drug conjugation ratio (DAR) × toxin molecular weight; percentage of free small molecule drug release (%) = Ctoxin at each time point / Ctotal (toxin) × 100%. Concentration data at each time point are retained to 3 significant figures, and the mean, standard deviation, and release percentage are retained to 2 decimal places.

[0445] 4. The results, summarizing the concentrations and release percentages of toxins in plasma from different species at each time point, are shown in Table 27 below.

[0446] The results showed that after incubation at 37°C for 504 h, the release percentages of toxin from PBS solution, cynomolgus monkey plasma, and human plasma using 45A5G10-HZ-B81 (DAR8) were 0.58%, 0.44%, and 0.38%, respectively. With increasing incubation time, no significant increase was observed in the release of toxins from any of the tested matrix components (<0.6%), confirming that the ADC exhibits good plasma and circulatory stability. Other embodiments of the ADC in this application also showed good plasma and circulatory stability in the same testing experiments, for example, with a toxin release percentage of less than 1%.

[0447] Although specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings published, and such changes are all within the scope of protection of this disclosure. The scope of protection of this disclosure is given by the appended claims and any equivalents thereof.

Claims

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 heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 16, HCDR2 as shown in SEQ ID NO: 18, and HCDR3 as shown in SEQ ID NO: 19; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 23, LCDR2 as shown in SEQ ID NO: 24, and LCDR3 as shown in SEQ ID NO: 22; wherein, As described above, HCDR1-3 and LCDR1-3 are determined according to the Kabat definition scheme; 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 anti-c-MET antibody or its antigen-binding fragment as claimed in claim 1, wherein the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:

9.

3. The anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2, further comprising a heavy chain constant region and a light chain constant region of the antibody.

4. The anti-c-MET antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is selected from the constant regions of human antibody κ or λ chains.

5. The anti-c-MET antibody or its antigen-binding fragment as described in any one of claims 1-4, wherein the antibody comprises a heavy chain constant region as shown in SEQ ID NO:51 and a light chain constant region as shown in SEQ ID NO:

52.

6. The anti-c-MET antibody or its antigen-binding fragment as claimed in claim 1 or 2, comprising: 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.

7. The anti-c-MET antibody or its antigen-binding fragment as claimed in claim 6, wherein the anti-c-MET antibody comprises: a heavy chain containing an amino acid sequence as shown in SEQ ID NO:56 or composed of said amino acid sequence and a light chain containing an amino acid sequence as shown in SEQ ID NO:57 or composed of said amino acid sequence.

8. A multispecific antibody comprising the anti-c-MET antibody of claim 1 or 2 or an antigen-binding fragment thereof.

9. The multispecific antibody of claim 8, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

10. A nucleic acid molecule encoding the anti-c-MET antibody of claim 1 or 2 or its antigen-binding fragment, or the multispecific antibody of claim 8 or 9.

11. A host cell comprising the nucleic acid molecule as described in claim 10.

12. A method for preparing an anti-c-MET antibody or an antigen-binding fragment thereof, or a multispecific antibody thereof, wherein the method comprises culturing a host cell as described in claim 11 under conditions suitable for expressing a nucleic acid encoding the anti-c-MET antibody or its antigen-binding fragment.

13. The method of claim 12, further comprising isolating the anti-c-MET antibody or its antigen-binding fragment, or its multispecific antibody.

14. An anti-c-MET antibody or an antigen-binding fragment thereof, or a multispecific antibody thereof, produced by the method of claim 12 or 13.

15. An immunoconjugate comprising: an anti-c-MET antibody as claimed in claim 1 or 2, or an antigen-binding fragment thereof, and an effector molecule, wherein the effector molecule is conjugated to the anti-c-MET antibody.

16. The immunoconjugate of claim 15, wherein the effector molecule is selected from antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

17. A method for in vivo and / or in vitro immunoassay or determination of c-MET, the method comprising the step of contacting a subject or a sample from a subject with an anti-c-MET antibody as described in claim 1 or 2.

18. An antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, comprising an anti-c-MET antibody as claimed in claim 1 or 2 or its antigen-binding fragment, or a multispecific antibody comprising an antibody or its antigen-binding fragment, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

19. The antibody-drug conjugate of claim 18, wherein the stereoisomer, the prodrug, the pharmaceutically acceptable salt, the tautomer, or the pharmaceutically acceptable solvate thereof, wherein the structure of the antibody-drug conjugate is as shown in formula (I): Equation (I) in which: Ab is the anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2, or a multispecific antibody containing the antibody or its antigen-binding fragment, wherein, preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; D is an active pharmaceutical ingredient; q is an integer selected from 1 to 20; L is a positional... and location The linking group is covalently linked to the antibody or its antigen-binding fragment Ab and the active pharmaceutical unit D, respectively; wherein position 1 is covalently linked to the antibody Ab or its antigen-binding fragment via an S atom, and position 2 is linked to D; and wherein the linking group has a structure selected from one of the following: Among them, R1 and R2 are independently selected from C 1-6 Alkyl groups and H.

20. The antibody-drug conjugate of claim 19, wherein its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

21. The antibody-drug conjugate of claim 19 or 20, wherein the stereoisomer, the prodrug, the pharmaceutically acceptable salt, the tautomer, or the pharmaceutically acceptable solvate thereof, wherein the active pharmaceutical unit is selected from DNA topoisomerase inhibitors or tubulin inhibitors.

22. The antibody-drug conjugate of claim 21, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the active pharmaceutical unit is a DNA topoisomerase inhibitor, and the DNA topoisomerase inhibitor is a camptothecin-type bioactive molecule.

23. The antibody-drug conjugate of claim 22, wherein its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the camptothecin-type bioactive molecule is selected from camptothecin, DXD, substituent-modified camptothecin or substituent-modified DXD, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, and rubotecan.

24. The antibody-drug conjugate of claim 21, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the active pharmaceutical unit is a tubulin inhibitor.

25. The antibody-drug conjugate of claim 24, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the tubulin inhibitor is selected from MMAF-type tubulin inhibitors and MMAE-type tubulin inhibitors.

26. An antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof, wherein the antibody-drug conjugate is a compound of formula (IIA-1), (IIA-2), (IIB-1), or (IIB-2): (IIA-1) (IIA-2) (IIB-1) (IIB-2) Wherein, Ab is the anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2, or a multispecific antibody comprising an antibody or its antigen-binding fragment, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; R1 and R2 are independently selected from C 1-6 Alkyl and H; D is 、 、 、 、 、 、 、 or ; and q is an integer selected from 1 to 20.

27. The antibody-drug conjugate of claim 19 or 26, wherein its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein R1 and R2 are independently selected from C 1-4 alkyl.

28. The antibody-drug conjugate of claim 26, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the antibody-drug conjugate has a structure selected from one of the following: in, q is an integer selected from 1 to 20; Ab is the anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2, or a multispecific antibody containing the antibody or its antigen-binding fragment, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

29. The antibody-drug conjugate of claim 28, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the Ab comprises: a heavy chain as shown in SEQ ID NO:56 and a light chain as shown in SEQ ID NO:

57.

30. An antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof, wherein the antibody-drug conjugate has the following structure: in, Ab is the anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2, or the multispecific antibody as described in claim 8 or 9, wherein Ab comprises a heavy chain variable region as shown in SEQ ID NO: 10 and a light chain variable region as shown in SEQ ID NO: 9; and q is an integer selected from 1 to 20.

31. The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate as claimed in claim 30, wherein the Ab comprises a heavy chain as shown in SEQ ID NO:56 and a light chain as shown in SEQ ID NO:

57.

32. The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate as described in claim 30, wherein, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

33. The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate as described in claim 32, wherein, q can be 2, 4, 6 or 8.

34. The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate as described in claim 32, wherein, q is 8.

35. A method for preparing an antibody-drug conjugate targeting c-MET, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof, wherein the antibody-drug conjugate has a structure as shown in Formula (I): Equation (I) in which: Ab is the anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2, or a multispecific antibody containing the antibody or its antigen-binding fragment, wherein, preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; D is an active pharmaceutical ingredient; q is an integer selected from 1 to 20; L is a positional... and location The linking group is covalently linked to the antibody or its antigen-binding fragment Ab and the active pharmaceutical unit D, respectively; wherein position 1 is covalently linked to the antibody Ab or its antigen-binding fragment via an S atom, and position 2 is linked to D; and wherein the linking group has a structure selected from one of the following: Among them, R1 and R2 are independently selected from C 1-6 Alkyl and H; wherein the method comprises the following steps: (1) reacting an anti-c-MET antibody or a fragment thereof with a reducing agent in a buffer to obtain a reduced antibody or a fragment thereof; and (2) crosslinking a linker-load (linker-drug conjugate) with the reduced antibody or a fragment thereof obtained in step (1) in a mixture of buffer and organic solvent to obtain an antibody-drug conjugate targeting c-MET.

36. The method of claim 35, wherein the reducing agent is a disulfide bond reducing agent.

37. The method of claim 35, wherein the buffer solution has a pH of 6.0-8.

0.

38. The method of claim 35, wherein the buffer solution is a phosphate buffer solution.

39. The method of claim 35, wherein the organic solvent is dimethyl sulfoxide.

40. An antibody-drug conjugate targeting c-MET, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof, produced by the method of claim 35.

41. A group of antibody-drug conjugates comprising, or consisting of, the antibody-drug conjugate as described in claim 18, 19, 26, 30 or 40, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or a pharmaceutically acceptable solvate thereof, wherein the antibody-drug conjugate has one, two or more q values.

42. The antibody-drug conjugate family of claim 41, wherein, The average DAR of the antibody-drug conjugate group is selected from an integer or decimal number between 1 and 16.

43. The antibody-drug conjugate group of claim 42, wherein the average DAR is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5 or 7.5-8.

5.

44. The antibody-drug conjugate family of claim 42, wherein, The average DAR of the antibody-drug conjugate group is selected from about 2.0, 4.0, 6.0 or 8.

0.

45. A pharmaceutical composition comprising: an anti-c-MET antibody as described in claim 1 or 2, or an antigen-binding fragment thereof; a multispecific antibody comprising an antibody or an antigen-binding fragment thereof, wherein preferably the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate comprising an antibody or its antigen-binding fragment, or a multispecific antibody; a nucleic acid molecule encoding an antibody or its antigen-binding fragment, or a multispecific antibody; an immunoconjugate comprising: an antibody or its antigen-binding fragment and an effector molecule, wherein the effector molecule is conjugated to the antibody; or a group of antibody-drug conjugates comprising or consisting of an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the antibody-drug conjugate has one, two, or more q values; and one or more pharmaceutically acceptable excipients.

46. ​​The anti-c-MET antibody or its antigen-binding fragment as described in claim 1 or 2; a multispecific antibody comprising an antibody or its antigen-binding fragment, wherein preferably the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody; an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate comprising an antibody or its antigen-binding fragment, or a multispecific antibody; a nucleic acid molecule encoding an antibody or its antigen-binding fragment, or a multispecific antibody; an immunoconjugate comprising an antibody or its antigen-binding fragment and an effector molecule. The use of the effector molecule coupled to the anti-c-MET antibody; an antibody-drug conjugate group comprising, or consisting of, an antibody-drug conjugate, 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 one, two or more q values; or a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, a multispecific antibody, an antibody-drug conjugate, a nucleic acid molecule, an immunoconjugate, an antibody-drug conjugate group and one or more pharmaceutically acceptable excipients, in the preparation of a medicament for the treatment or prevention of diseases related to c-MET activity.

47. The use as described in claim 46, wherein the disease associated with c-MET activity is a tumor associated with c-MET activity.

48. The use as described in claim 47, wherein the tumor is selected from: lung cancer, colon cancer, rectal cancer, gastric cancer, and colorectal cancer.

49. The use as described in claim 48, wherein the lung cancer is selected from: non-small cell lung cancer, small cell lung cancer, or lung adenocarcinoma.

50. The use as claimed in claim 48, wherein the colon cancer is human colonic adenocarcinoma, and wherein the colorectal cancer is colorectal adenocarcinoma.

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