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, and preparation methods and uses thereof Technical Field

[0001] The present application belongs to the field of medical technology and relates to various antibodies, antibody-drug conjugates and methods for preparing the same, as well as their use in preventing and / or treating diseases associated with abnormal cell activity, including but not limited to preventing and / or treating tumor diseases. Background Art

[0002] c-Met is a tyrosine kinase receptor expressed on the cell membrane. It binds to its ligand, HGF, through its Sema domain, triggering a downstream phosphorylation cascade, ultimately promoting cell proliferation. Currently, there are three main types of inhibitors targeting c-Met kinase: biological antagonists of HGF and c-Met, antibodies against HGF and c-Met, and small molecule c-Met inhibitors. Existing clinical results indicate that antibodies directly targeting HGF and c-Met, or small molecule inhibitors of c-Met, have been less than ideal in efficacy.

[0003] 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, the first anti-c-Met antibody developed, was derived from the humanization of the c-Met agonist antibody 5D5.

[0004] Cytotoxic chemotherapy was once the standard treatment for cancer, but highly potent cytotoxic molecules can kill normal cells and cause severe side effects. Targeted anti-tumor drugs, due to their combined targeting and anti-tumor activity, have become a hot topic in oncology research. However, these drugs often produce significant side effects due to their target selectivity, limiting their therapeutic efficacy. Biological macromolecule drugs, such as antibodies or antibody fragments, while highly targeted, have limited or no therapeutic effect against solid tumors. ADCs, conjugates of antibodies and small molecule drugs, combine the targeting properties of antibodies with the activity of bioactive molecules, creating a biological missile with highly promising efficacy and safety advantages. Antibodies guide ADCs to target cells, where they are then internalized. The small molecule drug is then released through enzymatic cleavage by specific enzymes to treat the disease.

[0005] Therefore, identifying a monoclonal antibody with high affinity and specific recognition for cMet, while effectively blocking HGF binding and downstream signaling pathways, is crucial for subsequent ADC drug development. Developing differentiated, higher-quality, and safer antibodies or ADCs targeting c-Met could provide cancer patients with a wider range of treatment options and hold broad market potential.

[0006] Summary of the Invention

[0007] The present disclosure provides an antibody or antigen-binding fragment thereof targeting c-MET, an antibody-drug conjugate, and their use in treating cancer. The present disclosure also provides nucleotides, polynucleotide combinations, expression vectors, and expression vector combinations encoding the above-mentioned c-MET antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising the above-mentioned c-MET antibodies or antigen-binding fragments thereof, and antibody-drug conjugates, as well as their use in preparing medicaments for treating or preventing cancer.

[0008] In a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof targeting c-MET, and specifically, provides an anti-c-MET antibody or antigen-binding fragment thereof. In some embodiments, the c-MET is human c-MET.

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

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

[0011] (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

[0012] (2) The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11.

[0013] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the present disclosure, HCDR1, HCDR2, and HCDR3 are selected from:

[0014] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12, or

[0015] (ii) a sequence comprising 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 relative to the sequence of any one of (i),

[0016] Preferably, the HCDRs are defined according to the AbM, Chothia, Kabat, Contact or IMGT definition schemes.

[0017] In some embodiments, the three complementarity determining regions (LCDRs) of the present disclosure, LCDR1, LCDR2, and LCDR3, from the light chain variable region are selected from

[0018] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11,

[0019] or

[0020] (ii) a sequence comprising 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 relative to the sequence of any one of (i),

[0021] Preferably, the LCDR is determined according to the AbM, Chothia, Kabat, Contact or IMGT definition scheme.

[0022] In some embodiments, the anti-C-MET antibody or antigen-binding fragment thereof as described in any of the above items comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:10; and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO:9.

[0023] In some embodiments, the anti-C-MET antibody of any of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:12; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:11.

[0024] In some embodiments, the anti-C-MET antibody of any of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:2; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:1.

[0025] In some embodiments, the anti-C-MET antibody of any of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:4; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:3.

[0026] In some embodiments, the anti-C-MET antibody of any of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:6; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:5.

[0027] In some embodiments, the anti-C-MET antibody of any of the above items comprises HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:8; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO:7.

[0028] In certain embodiments, HCDRs 1-3 and LCDRs 1-3 are identified using different assays or systems or defined schemes.

[0029] In certain embodiments, HCDR1-3 and LCDR1-3 are identified according to the AbM, Chothia, Kabat, Contact or IMGT definition schemes. In certain embodiments, the complementarity determining regions HCDR1-3 and LCDR1-3 are determined based on the corresponding heavy chain and light chain variable regions according to the Chothia, Kabat or IMGT definition schemes, as shown in the sequence and detailed information table.

[0030] In some embodiments, the HCDR1 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

[0031] In some embodiments, the HCDR2 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

[0032] In some embodiments, the HCDR2 determined based on the IMGT, Kabat or Chothia definition scheme comprises the amino acid sequence shown in SEQ ID NO: 38 or 61, or consists of the amino acid sequence, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 38 or 61.

[0033] In some embodiments, the HCDR3 determined based on the IMGT, Kabat or Chothia definition scheme comprises, or consists of, the amino acid sequence shown in the following SEQ ID NO, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

[0034] In some embodiments, LCDR1 determined based on the IMGT, Kabat or Chothia definition scheme comprises or consists of the amino acid sequence shown in the following SEQ ID NO, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

[0035] In some embodiments, the LCDR2 determined based on the IMGT, Kabat or Chothia definition scheme comprises or consists of the amino acid sequence shown in the following SEQ ID NO, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

[0036] In some embodiments, the LCDR3 determined based on the IMGT, Kabat or Chothia definition scheme comprises or consists of the amino acid sequence shown in the following SEQ ID NO, or comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in the following SEQ ID NO:

[0037] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0038] a. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively;

[0039] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the IMGT definition scheme.

[0040] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0041] b. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 38 (QIRLKSLNYATHYAXSVKG, wherein X may be any amino acid, such as E or Q), and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising, or consisting of, the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively;

[0042] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0043] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0044] c. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively;

[0045] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0046] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0047] d. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively;

[0048] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0049] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0050] e. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 53, and SEQ ID NO: 19, respectively; the light chain variable region comprises, or consists of, the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively;

[0051] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Chothia definition scheme.

[0052] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0053] f. the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively;

[0054] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the IMGT definition scheme.

[0055] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0056] 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 respectively consist 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: 37 and SEQ ID NO: 35, or respectively consist of the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 35. NO:37 and the amino acid sequence shown in SEQ ID NO:35;

[0057] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0058] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0059] h. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively;

[0060] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0061] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0062] i. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 32, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively;

[0063] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0064] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0065] j. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 32, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively;

[0066] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Chothia definition scheme.

[0067] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0068] k. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively;

[0069] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the IMGT definition scheme.

[0070] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0071] 1. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively;

[0072] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Kabat definition scheme.

[0073] In some embodiments, the anti-C-MET antibody of any of the above comprises a heavy chain variable region and / or a light chain variable region, wherein:

[0074] m. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 44, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise, or consist of, the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively;

[0075] Preferably, the HCDR1-3 and LCDR1-3 described above are defined according to the Chothia definition scheme.

[0076] In some embodiments, the three heavy chain CDRs comprised by the anti-C-MET antibodies or antigen-binding fragments thereof involved in the present disclosure contain no more than five amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions) in total compared to each of the above-mentioned groups of HCDR1-3. In some embodiments, the three light chain CDRs comprised by the anti-C-MET antibodies or antigen-binding fragments thereof involved in the present disclosure contain no more than five amino acid changes (e.g., amino acid substitutions, preferably conservative substitutions) in total compared to each of the above-mentioned groups of LCDR1-3.

[0077] The anti-C-MET antibody or antigen-binding fragment thereof as described in any of the above items 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 comprised in the heavy chain variable region and the light chain variable region undergo 1, 2, or 3 amino acid substitutions. In some preferred embodiments, the substitutions are conservative substitutions.

[0078] In some embodiments, the anti-C-MET antibody as described in any of the above items is a humanized antibody, a murine antibody or a chimeric antibody. In some embodiments, the antibodies of the present disclosure are humanized. Humanization can be achieved by replacing one or more amino acid residues in the heavy chain variable region and the light chain variable region of a natural antibody of non-human origin, especially the framework region sequence, with residues at the corresponding positions in the variable region of a conventional antibody from a human. Methods for humanizing antibodies are well known in the art. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of the antibody. Tests for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0079] In some embodiments, the anti-c-MET antibody as described in any of the above items 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).

[0080] In some embodiments, the heavy chain variable region of the present disclosure is

[0081] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10 or 12; or

[0082] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10 or 12; or

[0083] (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, 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 regions.

[0084] In some embodiments, the light chain variable region of the present disclosure is

[0085] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9 or 11; or

[0086] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9 or 11; or

[0087] (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, 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 regions.

[0088] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO: 9.

[0089] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 9.

[0090] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO: 11.

[0091] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 12; and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 11.

[0092] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO: 1.

[0093] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0094] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO:3.

[0095] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4; and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:3.

[0096] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO:5.

[0097] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:6; and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:5.

[0098] In some embodiments, the anti-C-MET antibody of any of the above 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 to 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 to SEQ ID NO: 7.

[0099] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain variable region and / or a light chain variable region, wherein: the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:8; and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:7.

[0100] In some embodiments, the anti-C-MET antibody as described in any of the above further comprises an antibody heavy chain constant region. In some embodiments, the anti-C-MET antibody as described in any of the above further comprises an antibody light chain constant region. In some embodiments, the anti-C-MET antibody as described in any of the above further comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3, or IgG4 constant regions. In some embodiments, the light chain constant region is selected from human antibody kappa or lambda chain constant regions.

[0101] In some preferred embodiments, the antibody heavy chain constant region of the present disclosure:

[0102] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 51;

[0103] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 51; or

[0104] (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: 51.

[0105] In some embodiments, the amino acid changes occur in the Fc region.

[0106] In some embodiments, the light chain constant region of an antibody of the present disclosure:

[0107] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 52;

[0108] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 52; or

[0109] (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.

[0110] In some embodiments, the antibody comprises a heavy chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:51 and a light chain constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:52.

[0111] In some embodiments, the anti-C-MET antibody as described in any of the above further comprises an antibody heavy chain. In some embodiments, the anti-C-MET antibody as described in any of the above further comprises an antibody light chain. In some embodiments, the anti-C-MET antibody as described in any of the above further comprises an antibody heavy chain and a light chain.

[0112] In some preferred embodiments, the antibody heavy chains of the present disclosure:

[0113] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 56 or 58;

[0114] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 56 or 58; or

[0115] (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 an amino acid sequence selected from SEQ ID NO: 56 or 58.

[0116] In some embodiments, the antibody light chain of the present disclosure:

[0117] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 57 or 59;

[0118] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 57 or 59; or

[0119] (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 selected from SEQ ID NO: 57 or 59.

[0120] In some embodiments, the anti-C-MET antibody of any of the above comprises:

[0121] A heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO:57.

[0122] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 57.

[0123] In some embodiments, the anti-C-MET antibody of any of the above comprises:

[0124] A heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO:59.

[0125] In some embodiments, the anti-C-MET antibody of any of the above items comprises a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 58, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 59.

[0126] In some embodiments, the present disclosure also provides an anti-C-MET antibody or antigen-binding fragment thereof, wherein the antibody competes with the anti-C-MET antibody as described in any of the preceding items for binding to human C-MET. As defined herein, an antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays, biointerferometry (e.g., Fortebio), or surface plasmon resonance (Biacore).

[0127] In some embodiments, the anti-C-MET antibody or antigen-binding fragment thereof competes with the 45A5G10-Hz antibody for binding to human C-MET.

[0128] In some embodiments, the anti-C-MET antibody or antigen-binding fragment thereof competes with the 55A10G6-Hz antibody for binding to human C-MET.

[0129] In some embodiments, the anti-C-MET antibody or antigen-binding fragment thereof competes for binding to human C-MET simultaneously with the 55A10G6-Hz antibody and the 45A5G10-Hz antibody.

[0130] In one embodiment of the present disclosure, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In some embodiments, the amino acid changes are conservative changes. For polypeptide sequences, "conservative changes" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence.

[0131] Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. A conservative substitution refers to the replacement of one amino acid with another within the same class, such as an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists eight groups of amino acids containing conservative substitutions for each other: 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 embodiments, the term "conservative sequence changes" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the disclosed antibody molecule or binding protein molecule containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the target antigen relative to the parent antibody or binding protein.

[0132] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0133] In certain embodiments, substitution occurs in the CDR regions of an antibody. Typically, the variants obtained have modifications (e.g., improvements) relative to certain biological properties of the parent antibody (e.g., increased affinity) and / or will have certain biological properties that are substantially retained of the parent antibody.

[0134] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0135] In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties that are known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.

[0136] In certain embodiments, the antibodies provided by the present disclosure are multispecific antibodies, such as bispecific antibodies, trispecific antibodies, or tetraspecific antibodies.

[0137] In a second aspect, the present disclosure provides a multispecific binding molecule, such as a multispecific antibody, comprising the anti-C-MET antibody or antigen-binding fragment thereof described above. In certain preferred embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0138] In a third aspect, in some embodiments, the present disclosure further provides a nucleic acid molecule encoding the anti-C-MET antibody or fragment thereof or any heavy chain or light chain thereof as described in any of the preceding items.

[0139] For example, the nucleic acids disclosed herein include nucleic acids encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, and 56-59, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, and 56-59. As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding the molecules disclosed herein can be generated using methods well known in the art, such as de novo solid phase DNA synthesis or PCR amplification.

[0140] The heavy chain and / or light chain of the antibody molecule disclosed herein may be fused to the N-terminus with a secretory signal peptide and / or a tag peptide that facilitates purification for production and purification.

[0141] The present disclosure also relates to vectors comprising the nucleic acid, such as expression vectors, such as eukaryotic expression vectors. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pTT5 vector, such as pTT5-mFc and pTT5-hFc vectors.

[0142] The present disclosure also provides a host cell comprising a nucleic acid molecule or vector as described in any of the preceding items. 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, for example, a bacterium, such as Escherichia coli.

[0143] The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0144] In a fourth aspect, the present disclosure provides a method for preparing an anti-c-MET antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or fragment thereof (preferably an antigen-binding fragment) or either one or both chains thereof, and optionally isolating the antibody or fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-c-MET antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

[0145] Antibodies prepared as described herein can be purified by known 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 particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0146] In a fifth aspect, in some embodiments, the present disclosure also provides an immunoconjugate (e.g., an antibody-drug conjugate), comprising an anti-C-MET antibody or an antigen-binding fragment thereof as described in any of the preceding items, and an effector molecule, wherein the effector molecule is conjugated to the anti-C-MET antibody or an antigen-binding fragment thereof; preferably, the effector molecule is selected from radioactive isotopes, anti-tumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0147] In a sixth aspect, in some embodiments, the present disclosure also provides methods for using the antibodies or antigen-binding fragments thereof of the present invention for diagnosis and detection, and compositions for diagnosis and detection comprising the same.

[0148] In certain embodiments, any of the anti-c-MET antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of c-MET in a biological sample.

[0149] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is other liquid samples of blood, serum, or biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from tumor tissue or cancer tissue.

[0150] In one embodiment, an anti-c-MET antibody for use in a method of diagnosis or detection is provided.

[0151] In another embodiment, a method for detecting the presence of c-MET in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of c-MET protein in a biological sample. In certain embodiments, c-MET is human c-MET or cynomolgus monkey c-MET. In certain embodiments, the method comprises 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 a complex indicates the presence of c-MET. The method can be an in vitro or in vivo method. In one embodiment, the anti-c-MET antibody is used to select a subject suitable for treatment with the anti-c-MET antibody, for example, where c-MET is a biomarker for selecting the subject. In some embodiments, the method is performed in vitro or in vivo.

[0152] In certain embodiments, labeled antibodies or fragments thereof are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected moieties, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions.

[0153] In some embodiments provided herein, the sample is obtained prior to treatment with an antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained prior to treatment with an alternative therapy. In some embodiments, the sample is obtained during treatment with an alternative therapy, or after treatment with an alternative therapy.

[0154] In some embodiments, c-MET is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0155] In some embodiments, a method for treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject 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 of the present invention, or an antibody-drug conjugate, pharmaceutical composition, formulation, combination product, etc. comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.

[0156] Therefore, in one embodiment, the present disclosure also provides a method for immunodetection or determination of C-MET, comprising the step of contacting a subject or a sample from a subject with the anti-C-MET antibody as described in any of the preceding items.

[0157] In a seventh aspect, the present disclosure further provides an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof, which contains the antibody or antigen-binding fragment thereof described in any one of the foregoing.

[0158] In some embodiments, the structure of the antibody drug conjugate is as shown in formula (I):

[0159] in:

[0160] Ab is any of the aforementioned antibodies or antigen-binding fragments thereof;

[0161] D is the active drug unit;

[0162] L is a linker, which is covalently linked to the antibody or its antigen-binding fragment Ab and the active drug unit D respectively;

[0163] q is an integer selected from 1-20.

[0164] In some embodiments, q is selected from 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 2-8, or 4-6; for example, an integer selected from 1-10.

[0165] Preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0166] In some embodiments, the L is covalently linked to an amino residue or a sulfhydryl residue on the antibody Ab; preferably, the L is covalently linked to a sulfhydryl residue on the antibody Ab; more preferably, the L is covalently linked to a sulfhydryl residue formed after the interchain disulfide bond on the antibody Ab is opened.

[0167] In some embodiments, the L is a cleavable linker or a non-cleavable linker. Preferably, the L is a cleavable linker.

[0168] In some embodiments, the cleavable linker comprises a peptide unit, wherein the peptide unit comprises 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 stereoisomers thereof;

[0169] In some embodiments, the peptide unit is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide comprising at least one (e.g., one, two or three) amino acid residue represented by AA1 or a stereoisomer thereof.

[0170] In some preferred embodiments, the peptide unit is a dipeptide, tripeptide or tetrapeptide comprising one amino acid residue represented by AA1 or a stereoisomer thereof.

[0171] In some embodiments, the peptide unit consists of the following amino acids:

[0172] i. at least one (eg, one, two, or three) amino acid residue represented by AA1 or a stereoisomer thereof, and,

[0173] ii. at least one natural amino acid residue and / or at least one non-natural amino acid residue.

[0174] AA 1 The structures of the indicated amino acid residues are shown below,

[0175] in:

[0176] R a 、R b In, either one is H and the other is r 1 is 4;

[0177] Or, R a With R b Together with the carbon atoms they are connected to, they form the 0 substituted 5-6 membered heterocycle;

[0178] R m1 、R n1 are each independently selected from hydrogen, C1-6 Alkyl and C 3-6 Cycloalkyl;

[0179] R 0 Selected from C 1-6 Alkyl, C 3-6 Cycloalkyl, -NR m2 R n2 and optionally C 1-6 an alkyl-substituted 5-6 membered heterocyclic group;

[0180] R m2 、R n2 are each independently selected from hydrogen and C 1-6 alkyl.

[0181] Preferably, the amino acid residue is selected from -Val-, -Ala-, -Gly-, -Cit-, -AA 1 -, -Arg-, -Phe-, -Lys-, and -Asn-.

[0182] Preferably, the peptide unit is selected from the group consisting of -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-Ala-(-Gly-Gly-Val-Ala-).

[0183] In some preferred embodiments, said L is

[0184] in,

[0185] L1 is selected from:

[0186] Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C 6-10Aryl, 5-10 membered heteroaryl and amide (preferably selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond); Rx, Ry are independently selected from H and C 1-4 Alkyl; 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 connected to the antibody or antigen-binding fragment thereof through an S atom, and position 2 is connected to L2 or L3;

[0187] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0188] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0189] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0190] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0191] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3.

[0192] L2 is absent or present. When L2 is present, L2 is selected from Each y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, the 1 position is connected to L1, and the 2 position is connected to L3;

[0193] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0194] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0195] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0196] In some embodiments, L2 is absent.

[0197] In some embodiments, L2 is selected from

[0198] L3 is selected from Position 1 is connected to L1 or L2, and position 2 is connected to L4 or D;

[0199] L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0200] In some preferred embodiments, the The structure is as follows:

[0201] Wherein, 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 antigen-binding fragment thereof through the S atom, and the 2 position is linked to D.

[0202] Preferably, the The structure is as follows:

[0203] Among them, position 1 is connected to the antibody or its antigen-binding fragment through the S atom, and position 2 is connected to D.

[0204] In some embodiments, the active drug unit is selected from a cytotoxic agent. In some embodiments, the active drug unit is selected from a DNA topoisomerase inhibitor (e.g., a camptothecin bioactive molecule, such as camptothecin, DXD, a substituent-modified camptothecin, or a substituent-modified DXD, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan) or a tubulin inhibitor (e.g., MMAF tubulin inhibitor, MMAE tubulin inhibitor).

[0205] In some embodiments, the antibody drug conjugate is a compound of Formula (IIA-1) or Formula (IIA-2):

[0206] in,

[0207] Ab is the above-mentioned anti-c-Met antibody or antigen-binding fragment thereof, or multispecific antibody;

[0208] R1 and R2 are independently selected from C 1-6 Alkyl and H; preferably C 1-4 alkyl;

[0209] D is

[0210] q is as defined above. In some preferred embodiments, q is 2, 4, 6 or 8.

[0211] In some embodiments, the antibody drug conjugate is a compound of formula (IIB-1) or formula (IIB-2):

[0212] in,

[0213] Ab is the above-mentioned anti-c-Met antibody or an antigen-binding fragment thereof, for example, a multispecific antibody or an antigen-binding fragment thereof;

[0214] R1 and R2 are independently selected from C 1-6 Alkyl and H; preferably C 1-4 alkyl;

[0215] D is

[0216] q is as defined above. In some preferred embodiments, q is 2, 4, 6 or 8.

[0217] In some embodiments, the antibody drug conjugate has the following structure:

[0218] wherein Ab and q are as defined above.

[0219] In an eighth aspect, the present disclosure provides a method for preparing an antibody-drug conjugate targeting C-MET, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof, wherein the antibody-drug conjugate has a structure shown in Formula I, comprising the following steps:

[0220] (1) reacting an anti-c-MET antibody or fragment thereof with a reducing agent in a buffer to obtain a reduced antibody or fragment thereof; preferably, the reducing agent is a disulfide bond reducing agent, such as TCEP; preferably, the buffer has a pH of 6.0-8.0, such as 6.5, 7.0, 7.5 or 8.0, more preferably a phosphate buffer;

[0221] (2) cross-linking the drug linker (linker-drug conjugate) with the reduced antibody or fragment thereof obtained in step (1) in a mixture of a buffer and an organic solvent to obtain an antibody drug conjugate targeting c-MET, wherein the buffer is as defined above. Preferably, the organic solvent is selected from dimethyl sulfoxide.

[0222] In some embodiments of the present disclosure, the c-MET antibody or fragment thereof is as defined above; the drug linker has a structure as shown in the following formula (IIIA-1), (IIIA-2), (IIIB-1) or (IIIB-2):

[0223] in,

[0224] R1 and R2 are independently selected from C 1-6 Alkyl, H; preferably C 1-4 alkyl;

[0225] D is

[0226] In some embodiments, the drug linker has the following structure:

[0227] The drug linker (Linker-Payload) disclosed herein can be prepared by various methods known in the art, such as by chemical synthesis. The linker-payload in the above-mentioned antibody-drug conjugate can be prepared with reference to WO22022170971 and then conjugated with the antibody to form an ADC.

[0228] In the ninth aspect of the present disclosure, the present disclosure provides a group of antibody-drug conjugates, comprising or consisting of the antibody-drug conjugates, stereoisomers, prodrugs, pharmaceutically acceptable salts, tautomers, or pharmaceutically acceptable solvates thereof according to the seventh aspect, wherein the antibody-drug conjugates have one, two or more q values.

[0229] In some embodiments, when one q value of the antibody drug conjugates in the population of antibody-drug conjugates accounts for the majority (e.g., 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99%), the q value and the average DAR are close.

[0230] In some embodiments, when there is only one q-value antibody drug conjugate in the population of antibody-drug conjugates, the q-value and the average DAR are equal.

[0231] In some embodiments, when the antibody drug conjugates of the antibody-drug conjugate population have two or more q values, the proportion of antibody drug conjugates with a particular q value in all antibody drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0232] In some embodiments, the average drug to antibody ratio (average DAR) in the antibody drug conjugates in the population of antibody-drug conjugates is an integer or decimal selected from 1-16, preferably 1-10.

[0233] In some embodiments, the average drug to antibody ratio (average DAR) of the population of antibody-drug conjugates is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5;

[0234] In some embodiments, the average drug to antibody ratio (average DAR) of the population of antibody-drug conjugates is selected from about 2.0, 4.0, 6.0, or 8.0;

[0235] In some embodiments, the average drug to antibody ratio (average DAR) in the antibody drug conjugates in the population of antibody-drug conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0236] In some embodiments, the antibody-drug conjugate population contains ADCs with a distribution of DARs of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 drug loading species). It is worth noting that degradation products can be produced such that the mixture may also contain DARs of 1, 3, 5, and 7. In addition, the antibody-drug conjugate population may also have an average DAR greater than 8. The antibody-drug conjugate is produced by reducing the interchain disulfide followed by conjugation. In some embodiments, the antibody-drug conjugate comprises both: an antibody-drug conjugate having a DAR of 4 or less (i.e., a drug loading species of 4 or less) and an antibody-drug conjugate having a DAR of 6 or more (i.e., a drug loading species of 6 or more).

[0237] In the tenth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the antibody-drug conjugate of the seventh aspect or its stereoisomer, prodrug, pharmaceutically acceptable salt, tautomer or pharmaceutically acceptable solvate thereof, the nucleic acid of the third aspect, the immunoconjugate of the fifth aspect or the antibody-drug conjugate group of the ninth aspect, and optionally one or more pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0238] In certain embodiments, the pharmaceutical composition comprises an effective amount of the following: the antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the stereoisomer-drug conjugate of the seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers or its pharmaceutically acceptable solvates, the nucleic acid of the third aspect, the immunoconjugate of the fifth aspect, or the antibody-drug conjugate group of the ninth aspect.

[0239] In certain embodiments, the pharmaceutical composition comprises the above-mentioned antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, and pharmaceutical excipients.

[0240] In certain embodiments, the pharmaceutical composition disclosed herein comprises the antibody-drug conjugate population of the ninth aspect, and pharmaceutically acceptable excipients.

[0241] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the anti-c-MET antibody or antigen-binding fragment thereof according to the first aspect of the present disclosure, and a pharmaceutically acceptable excipient.

[0242] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises the isolated nucleic acid molecule or vector as described above.

[0243] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the multispecific antibody of the second aspect of the present disclosure, and a pharmaceutically acceptable excipient.

[0244] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or antibody-drug conjugate population is an integer or decimal selected from 1-10.

[0245] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody-drug conjugates is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5;

[0246] In some embodiments, the DAR in the pharmaceutical composition or antibody-drug conjugate population is selected from the group consisting of: 2±0.5, 4±0.5, 5±0.5, 6±0.5, 7±0.5, 8±0.5;

[0247] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody-drug conjugates is selected from about 2.0, 4.0, 6.0, or 8.0;

[0248] In some embodiments, the drug to antibody ratio (average DAR) in the pharmaceutical composition or population of antibody-drug conjugates is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0249] In some embodiments, the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, or the drug conjugate of the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof and a buffer.

[0250] In some embodiments, the pharmaceutical composition comprises the seventh aspect drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof, and a buffer.

[0251] In some preferred embodiments, the buffer is selected from the group consisting of: histidine buffer, phosphate buffer, in some preferred embodiments, the buffer is selected from the group consisting of histidine buffer, in some preferred embodiments, the buffer is selected from the group consisting of 20 mM histidine buffer.

[0252] In an eleventh aspect, the present disclosure provides a use of an antibody or antigen-binding fragment thereof in the preparation of a kit for detecting the presence or level of c-MET in a sample. In another aspect, the present disclosure provides a diagnostic or therapeutic kit comprising one or more of the following substances: an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, an antibody-drug conjugate, an antibody-drug conjugate group, or a pharmaceutical composition as described herein. Optionally, the diagnostic or therapeutic kit further comprises instructions for use. In some embodiments, the kit is suitable for use in the diagnostic or detection methods described herein. In some embodiments, the kit is suitable for use in the treatment methods described herein.

[0253] In a twelfth aspect, the present disclosure provides the use of the disclosed substances (including the aforementioned antibody-drug conjugate composition or the aforementioned pharmaceutical composition) in the preparation of a medicament for treating and / or preventing diseases associated with abnormal cell 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.

[0254] In some embodiments, provided is a use of the anti-C-MET antibody or antigen-binding fragment thereof, 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.

[0255] In some embodiments, provided is a use of the anti-C-MET antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell antibody-drug conjugate, or multispecific antibody disclosed herein in the preparation of a medicament for treating or preventing a disease associated with the activity of C-MET or a disease associated with a target of C-MET.

[0256] In some embodiments, the anti-C-MET antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, antibody-drug conjugate, or multispecific antibody disclosed herein is used in the preparation of a drug for treating or preventing a tumor associated with the activity of C-MET.

[0257] In some embodiments, there is provided use of a substance of the disclosure in the preparation of a medicament for treating or preventing a disease associated with the activity of C-MET or a disease associated with a target of C-MET, wherein the substance of the disclosure is selected from the above-mentioned antibody-drug conjugate disclosed herein, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers, or its pharmaceutically acceptable solvates, the anti-C-MET antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the vector 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.

[0258] In some embodiments, provided is the use of the antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the antibody-drug conjugate, stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable salts thereof, tautomers thereof, or pharmaceutically acceptable solvates thereof of the seventh aspect, the antibody-drug conjugate group of the ninth aspect, or the pharmaceutical composition of the tenth aspect of the present disclosure in the preparation of a medicament for treating or preventing a disease associated with the activity of C-MET or a disease associated with a target of C-MET.

[0259] In a thirteenth aspect, the present disclosure provides a method for treating and / or preventing diseases associated with abnormal cell activity (e.g., tumors) by using the antibody or antigen-binding fragment thereof of the first aspect, the multispecific antibody of the second aspect, the antibody-drug conjugate of the seventh aspect, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers, or its pharmaceutically acceptable solvates, the nucleic acid of the third aspect, the vector 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.

[0260] In the twelfth and thirteenth aspects, the disease associated with C-MET activity, the disease associated with C-MET targets, or the disease associated with abnormal cell activity includes a tumor, such as cancer. The cancer may be in the early, middle, or late stages, or may be metastatic. In some embodiments, the cancer may be a solid tumor or a hematologic tumor.

[0261] In one embodiment, the tumor refers to elevated protein levels (e.g., expression) of c-MET, or elevated nucleic acid levels of c-MET in tumor tissue or tumor cells of the individual, e.g., compared to adjacent normal tissue or normal cells (e.g., normal cells in a tissue) of the individual, or the same tissue or cells therein of a healthy individual.

[0262] The tumor is selected from, but not limited to, lung cancer (such as non-small cell lung cancer, small cell lung cancer or lung adenocarcinoma), colon cancer (such as human colon adenocarcinoma), rectal cancer, gastric cancer, colorectal cancer (such as colorectal adenocarcinoma).

[0263] In the above-mentioned twelfth and thirteenth aspects, 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 vector 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 treatment modalities or therapeutic agents for the treatment of related diseases or for related uses.

[0264] In some embodiments, the treatment modality is surgery or radiation therapy.

[0265] In a fourteenth aspect, the present disclosure also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-c-MET antibody or fragment thereof (preferably an antigen-binding fragment) of the present invention, or an antibody-drug conjugate thereof, and one or more other therapeutic agents.

[0266] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0267] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0268] - a first container containing a pharmaceutical composition comprising an anti-c-MET antibody or a fragment thereof;

[0269] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0270] In a fifteenth aspect, the present disclosure further provides use of the antibody or antigen-binding fragment thereof of the first aspect and the multispecific antibody of the second aspect for preparing an antibody-drug conjugate.

[0271] In some embodiments, the antibody drug conjugate is selected from the antibody drug conjugate of the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a pharmaceutically acceptable solvate thereof.

[0272] The immunoglobulin molecules disclosed herein can be of 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, VH CDR (herein often represented by HCDR), VL domain, or VL CDR (herein often represented by LCDR) having any of the amino acid sequences described in the Sequence and Detailed Information Tables, or a fragment or variant thereof.

[0273] Preferably, the antibodies disclosed herein comprise or consist of a VH domain, a VH CDR (herein often represented by HCDR), a VL domain, or a VL CDR (herein often represented by LCDR) having any of the amino acid sequences described in the Sequence and Specific Information Tables, or a fragment or variant thereof.

[0274] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. The modifier "monoclonal" herein indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies and is not to be construed as requiring production by a particular method.

[0275] In some embodiments of the present disclosure, monoclonal antibodies also 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, and the remaining portion is identical or homologous to another type, class, or subclass of antibody, as long as they have the desired biological activity. Chimeric antibodies that can be used in the present disclosure include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0276] 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 complementary determining region fragments, diabodies, linear antibodies and single-chain antibody molecules. The term "antigen-binding fragment" as used herein refers to a partial fragment of an antibody with antigen-binding activity, wherein the fragment has all or part of the function of an 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 to the antigen. In addition, these functional fragments include not only fragments obtained by treating the full-length molecule of the antibody protein with an appropriate enzyme, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0277] The term "Fab'" as used herein 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, the Fab' disclosed herein also includes Fab' produced using genetically modified antibody genes.

[0278] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are linked by a linker or directly (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0279] The terms "variable region" or "variable domain" or "variable domain" refer to the domains of an antibody's heavy and / or light chains involved in antigen binding. Natural IgG antibodies, VH and VL, each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The terms "complementarity-determining regions" or "CDRs" refer to the regions within the variable domain that primarily contribute to antigen binding; "framework" or "FRs" refers to the variable domain residues excluding the CDR residues. VHs contain three CDR regions: HCDR1, HCDR2, and HCDR3; VLs contain three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to 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.

[0280] The boundaries of the amino acid sequence of the CDR can be determined by various well-known definition schemes, for example: the "Kabat" definition scheme rules (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" definition scheme, the "ABM" definition scheme, the "contact" definition scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and the ImMunoGenTics (IMGT) definition scheme (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between the various definition schemes is well known to those skilled in the art and is exemplified as shown below.

[0281] CDR defines the relationship between the schemes

[0282] As used herein, the antibody or antigen-binding fragment thereof still has the activity of binding to the antigen even if it contains variants, amino acid substitutions, deletions or additions.

[0283] The term "binding molecule" refers to any molecule that is capable of specifically binding to a target, such as an antibody or an antigen-binding fragment thereof, or a fusion protein. The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule, i.e., the molecule comprises 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 present invention also encompass multispecific molecules comprising multiple target binding regions, such as trispecific binding molecules. In some embodiments, the multispecific binding molecules of the present invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies.

[0284] The term "bispecific antibody," also known as a "bifunctional antibody conjugate," refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) linked by a coupling arm. This conjugate retains the activities of each antibody and thus possesses both bifunctionality and bispecificity. In one embodiment, provided herein is a bispecific antibody having binding specificity for c-Met and binding specificity for a second antigen.

[0285] The term "multispecific antibody" includes, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies, the former being antibodies with three different antigen-binding specificities, and the latter being antibodies with four different antigen-binding specificities. In some embodiments, the multispecific antibody has a binding specificity for c-Met and one or more binding specificities for other antigens.

[0286] The term "complete antibody" or "full-length antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. A complete antibody is preferably a complete antibody with one or more effector functions. In the present disclosure, a "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody comprising a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of a human immunoglobulin are also replaced with non-human residues. Moreover, humanized antibodies may also comprise residues that are not present in the recipient antibody or the donor antibody. These modifications are intended to further optimize the performance of the antibody. Humanized antibodies generally comprise at least one, usually two variable regions, in which all or nearly all of the hypervariable loops correspond to those of non-human immunoglobulins, while the FRs are entirely or almost entirely human immunoglobulin sequences. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc, usually a human immunoglobulin Fc).

[0287] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different antibody classes are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0288] In this article, the CDRs contained in the antibodies or antigen-binding fragments thereof disclosed herein can be determined according to various definition schemes known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof disclosed herein are preferably determined by the Kabat, Chothia or, AbM or IMGT definition schemes.

[0289] As used herein, the term "framework residue region" or "FR residues" refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0290] The compilation of the twenty conventional amino acids involved herein follows conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala; arginine can be represented by R or Arg; glycine can be represented by G or Gly; and glutamine can be represented by Q or Gln.

[0291] As used herein, the term "immunoconjugate" refers to an effector molecule connected to an antibody or its antigen-binding fragment via a linker, so that the antibody or its antigen-binding fragment can be used as a carrier to target and transport the effector molecule to the target site. The term "effector molecule" refers to the active portion of the antibody or antibody fragment of the present invention, and may include any portion for attaching the antibody or antibody fragment. In some embodiments, the effector molecule can be a drug such as a small molecule drug, DNA, RNA, enzyme or polypeptide, etc. In some embodiments, the immunoconjugate covers antibody drug conjugates (ADCs). Effector molecules or active portions suitable for being connected to antibodies include, for example, antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof. In some embodiments, the immunoconjugate of the present invention is an antibody drug conjugate, i.e., ADC.

[0292] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or regulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0293] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., tumor and infectious disease) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0294] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human. In certain embodiments, the subject (e.g., human) has a tumor and an infectious disease, or is at risk of having the above-mentioned disease.

[0295] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., tumors and infectious diseases) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., tumors and infectious diseases); an effective amount for treating a disease refers to 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 well within the capabilities of those skilled in the art. For example, an amount effective 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 condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0296] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0297] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0298] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit / test kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents with prophylactic or therapeutically effective levels in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0299] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (such as tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0300] The term "label" as used herein 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 reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0301] An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, 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).

[0302] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may comprise conservative changes.

[0303] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0304] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0305] As used herein, the terms "comprising" or "including" are intended to include the recited 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 indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0306] The term "pharmaceutically acceptable" means that when the molecule itself, molecule fragment or composition is appropriately administered to an animal or human, it does not produce adverse, allergic or other untoward reactions. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0307] The term "drug to antibody ratio" or "DAR" refers to the ratio of the number of drug moieties (D) coupled to an antibody portion (Ab) as described herein to the number of antibody portions. The DAR of an ADC can range from 1 to 20, but higher loadings are possible depending on the number of attachment sites on the antibody. The term DAR can 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. The DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio (molar ratio) of drug moieties (D) coupled to the Ab portion as described herein to the Ab portion in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the antibody drug conjugates of the present disclosure is 1.0-20.0, e.g., 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, e.g., 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, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 12.0, and 16.0, and ranges having two of these values ​​as endpoints.

[0308] In the process of determining the DAR value by mass spectrometry, the antibody has been reduced to separated heavy and light chains. DAR1 represents a conjugate containing a light chain or heavy chain coupled to one toxin molecule; DAR2 represents a conjugate containing a light chain or heavy chain coupled to two toxin molecules; and DAR3 represents a conjugate containing a light chain or heavy chain coupled to three toxin molecules.

[0309] The term "drug" refers to chemical substances that can alter or identify physiological functions and pathological states of the body and can be used to prevent, diagnose, and treat disease. This includes substances that inhibit or prevent cellular function and / or cause cell death or destruction. Drugs include cytotoxic agents, particularly small molecule cytotoxic agents. There is no strict distinction between drugs and poisons. Poisons are chemical substances that can have toxic effects on the body and harm human health even at relatively low doses. Excessive doses of any drug can produce toxic reactions.

[0310] Cytotoxic agents are substances that inhibit or prevent cell 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 cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic agents include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 13 1.I 125 、Y 90 、Re 186 、Re 188 、Sm 15 3. Bi 212 、P 32 and radioisotopes of Lu), chemotherapeutic drugs, antibiotics and nucleolytic enzymes. In addition, it includes but is not limited to DNA topoisomerase inhibitors (e.g., camptothecin-type bioactive molecules, such as camptothecin, DXD, camptothecin with modified substituents or DXD with modified substituents, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan) or microtubule inhibitors (e.g., MMAF-type microtubule inhibitors, MMAE-type microtubule inhibitors).

[0311] For the avoidance of doubt, the term "drug" that can be a component of an ADC does not refer solely to "drugs" approved by pharmaceutical regulatory authorities. It also includes any compound with potential therapeutic biological activity in clinical practice, R&D, or academic research. Furthermore, it should be understood that this term differs from the meaning of "drug for use in the preparation of a medicine." It should be understood that drug molecules may need to be functionalized or derivatized in order to be attached to a linker, and the resulting compounds are also included within the scope of the drugs disclosed herein.

[0312] In this disclosure, the "active drug unit" is the portion of an antibody-drug conjugate (ADC) other than the antibody and linker, which is derived from the drug defined above. For convenience, the "active drug unit" in the ADC of this disclosure may be referred to directly by the name of the drug, as understood by those skilled in the art.

[0313] As used in this disclosure, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the antibody drug conjugate or drug linker conjugate of this disclosure, and the salt is not biologically or otherwise undesirable. The conjugates of this disclosure (including antibody drug conjugates and drug linker conjugates) can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In this disclosure, pharmaceutically acceptable acid addition salts represent salts formed between the conjugates of this disclosure and organic or inorganic acids, 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 conjugates of the present 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; organic base salts, such as ammonium salts formed with organic bases containing N groups.

[0314] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0315] As used in this disclosure, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In a compound with one or more (e.g., one, two, three, or four) asymmetric centers, it can produce a racemic mixture, a single enantiomer, a diastereomeric mixture, and a separate diastereomer. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of this disclosure can exist as mixtures (commonly referred to as tautomers) of two or more structurally different forms in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this disclosure encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0316] Solid lines (-), solid wedges, or dashed wedges may be used in the present disclosure to depict carbon-carbon bonds of the compounds of the present disclosure. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom (e.g., specific enantiomers, racemic mixtures, etc.) are included. The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomers shown are present. Unless otherwise indicated, the compounds of the present disclosure are intended to exist in the form of stereoisomers (which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the present disclosure may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereoisomer pairs).

[0317] The present disclosure also includes all pharmaceutically acceptable isotopic compounds that are identical to the compounds of the present disclosure except that one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of suitable isotopes for inclusion in the compounds of the present disclosure include, but are not limited to, isotopes of hydrogen (e.g., 2 H. 3 H); carbon isotopes (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S).

[0318] The compounds of the present disclosure may exist in the form of solvates (preferably hydrates), wherein the compounds of the present disclosure contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of the polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0319] Also included within the scope of the present disclosure are metabolites of the compounds of the present disclosure, i.e., substances formed in vivo upon administration of the compounds of the present disclosure. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. of the administered compound. Thus, the present disclosure includes metabolites of the compounds of the present disclosure, including compounds produced by contacting the compounds of the present disclosure with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0320] The present disclosure further includes within its scope prodrugs of the compounds of the present disclosure. Typically, such prodrugs will be functional group derivatives of the compounds that are easily converted into the desired therapeutically active compound in vivo. Therefore, in these cases, the term "administering" for the methods of treatment disclosed herein should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but after administration to an individual, the prodrug forms are converted into the above-mentioned compounds in vivo. For example, in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985, conventional methods for selecting and preparing suitable prodrug derivatives are described.

[0321] For the purposes of this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These excipients are substances, other than active ingredients, that have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients or excipients also have important functions such as solubilization, dissolution assistance, and sustained-release control. They are important ingredients that may affect the quality, safety, and efficacy of drugs. Based on their source, they can be categorized as natural, semi-synthetic, and fully synthetic. Based on their functions and uses, pharmaceutical excipients can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. Based on their route of administration, pharmaceutical excipients can be divided into oral, parenteral, mucosal, transdermal or topical, nasal or oral inhalation, and ocular administration. The same pharmaceutical excipient or vehicle can be used in pharmaceutical preparations for different routes of administration and have different functions and uses. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0322] The pharmaceutical composition can be made into various suitable dosage forms according to the route of administration. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye preparations, pills, implants, aerosols, powder sprays, sprays, etc. Wherein, the pharmaceutical composition or suitable dosage form can contain 0.01mg to 1000mg of the compound of the present disclosure (including conjugates) or pharmaceutically acceptable salts thereof, preferably containing 0.1mg to 800mg, preferably containing 0.5-500mg, preferably containing 0.5 to 350mg, particularly preferably 1-250mg. It should be possible that the amount exceeding the above range is sometimes feasible.

[0323] The pharmaceutical composition can be administered in the form of an injection, including an injection solution, sterile powder for injection, and concentrated solution for injection. Usable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an infusion.

[0324] In the present 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 sulfhydryl group contained in the antibody itself after the disulfide bond is opened (for example, reduction of the disulfide bond by the reducing agent TCEP can open the disulfide bond to generate a sulfhydryl group -SH). In other words, the -S- between L and Ab is not an additional external sulfur atom.

[0325] As used herein, the terms "comprising" or "including" are intended 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 indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0326] As used herein, the term "linker" or "linker" refers to a segment that connects an active drug unit (drug molecule) to an antibody portion. In this regard, the linker, prior to attachment to the antibody or antigen-binding fragment thereof (i.e., a precursor linker), has functional groups that can form bonds with functional groups of the antibody or antigen-binding fragment thereof.

[0327] In the present disclosure, the term "antibody drug conjugate" or "ADC" refers to a substance obtained by connecting an active drug unit (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the active drug unit is connected to the targeting portion via a linker. The linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound (e.g., a c-Myc protein degrader) fragment from the targeting portion or the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the active drug unit is directly connected to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the active drug unit from the antibody or its antigen-binding fragment.

[0328] In this disclosure, the term "alkyl" refers to a linear or branched fully saturated hydrocarbon group that may be optionally substituted, preferably a C1-C 10 , more preferably C1-C8, C1-C6, or C1-C4 alkyl. Examples of alkyl 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, and the like.

[0329] In the present disclosure, the term "aryl" refers to a C6-C8 group having a single ring (e.g., phenyl) or a condensed ring (e.g., naphthyl, anthracenyl, phenanthrenyl, fluorenyl, etc.) which may be optionally substituted. 16 Aromatic hydrocarbon group, preferably C6-C 10 Aromatic hydrocarbon group.

[0330] In the present disclosure, the term "heteroaryl" refers to a 5-16 membered 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 membered aromatic group, more preferably a 5-6 membered aromatic group. Examples of heteroaryl groups include imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyrrolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, azaindolyl (e.g., 7-azaindolyl), benzimidazolyl, benzopyrazolyl, benzofuranyl, benzothienyl, benzothiazolyl, dibenzofuranyl, dibenzothienyl, quinolyl, isoquinolyl, naphthyridinyl, carbazolyl, azacarbazolyl (e.g., 1-azacarbazolyl, 2-azacarbazolyl, 1,8-diazacarbazolyl), indolizinyl, azaindolizinyl, phenoxazinyl, phenothiazinyl, and the like.

[0331] In this context, the term "3-6 membered cycloalkyl" or "C 3-6The term "cycloalkyl" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms, including cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexyl.

[0332] As used herein, the term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (at least one (e.g., 1, 2, or 3) of which is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom), including but not limited to pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, and the like.

[0333] As used herein, the term "5-6 membered heterocyclic group" refers to a cyclic group containing 5-6 ring atoms (wherein 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, piperazinyl, tetrahydropyranyl, and the like.

[0334] The term "antibody-drug conjugate population" refers to a group or mixture of the disclosed antibody-drug conjugates, their stereoisomers, prodrugs, pharmaceutically acceptable salts, tautomers, or pharmaceutically acceptable solvates thereof, wherein the q of the antibody-drug conjugates may be the same or different. Alternatively, this may be referred to as an "antibody-drug conjugate mixture."

[0335] Advantageous Effects of the Invention

[0336] In some embodiments, the antibodies or antigen-binding fragments thereof targeting c-MET developed in the present disclosure bind to c-Met with high affinity, have high affinity for human and monkey cMet, and have good binding specificity, and do not bind to rat or mouse cMet.

[0337] In some embodiments, the antibodies or antigen-binding fragments thereof targeting c-MET developed in the present disclosure have antagonistic effects, that is, antibodies that antagonize HGF-induced phosphorylation of c-Met and protein kinase B (PKB), rather than agonistic effects.

[0338] In some embodiments, compared to a control antibody, such as ABT700, the antibodies or antigen-binding fragments thereof developed in the present disclosure that target c-MET have efficient endocytosis activity, higher affinity, different binding epitopes, and the activity of competing with HGF ligands for binding to c-Met.

[0339] In some embodiments, the present disclosure provides anti-c-MET antibodies that are highly humanized and / or thermally stable, thereby being druggable and safely administered to human subjects without eliciting an immunogenic response.

[0340] Thus, in some embodiments, the c-MET-targeting antibodies or antigen-binding fragments thereof of the present disclosure exhibit specificity, reduced toxicity, stability, and enhanced physical and functional properties compared to known therapeutic agents.

[0341] At the same time, compared to the development of c-MET signal blocking inhibitors, which will only benefit a portion of the total number of tumor patients expressing c-MET, the c-MET ADC disclosed herein can overcome some limitations of signal blocking c-MET inhibitors, allowing more tumor patients with low c-MET expression to benefit.

[0342] In some embodiments, the disclosed antibody-drug conjugates (ADCs) achieve enrichment in the tumor microenvironment, unique in vivo enzymatic cleavage properties of the linker, and a conjugation method with the targeting moiety, combined with extensive in vivo and in vitro efficacy screening and validation, to obtain a novel class of antibody-based bioactive molecule conjugates. The conjugates obtained using the above-described methods can achieve a variety of the following surprising technical effects:

[0343] In some embodiments, the conjugate obtained according to the above method has better solubility and excellent chemical stability. For example, the reversible Michael addition reaction caused by the maleimide linkage in traditional ADCs does not occur, so a high DAR value can be obtained. In some embodiments, the DAR value of the conjugate can reach 6-8, or even higher.

[0344] Having extremely high coupling efficiency. In some embodiments, the coupling efficiency can reach or exceed 90%;

[0345] Therefore, in one embodiment, the present disclosure has discovered through extensive research a class of ADCs that have high plasma stability but can also be cleaved in the tumor microenvironment (both inside and outside tumor cells). This allows for release both inside tumor cells and in tumor tissues, maximizing the delivery of the ADC to tumor tissues and cells, thereby also exerting its therapeutic efficacy. Therefore, it can produce a good anti-tumor effect in tumors with low or no antigen expression. The ADCs disclosed herein have shown significant tumor inhibitory activity on tumor cells with different c-MET expression levels.

[0346] In some embodiments, after incubation at 37° C. for 504 hours, the ADC disclosed herein has a toxin release percentage of <0.6% in PBS solution, cynomolgus monkey plasma, and human plasma, respectively, showing excellent plasma stability and circulation stability. The conjugate (ADC) obtained according to the above method improves the exposure of the entire ADC molecule to the relatively acidic tumor environment by adjusting the physicochemical properties of the linker and the overall ADC molecule. As a result, the ADC has better tumor tissue targeting, i.e., the ability to be enriched in the tumor microenvironment, increases the ratio of the intratumoral and blood concentrations of the bioactive molecule, and reduces the mechanism-related toxicity of the ADC molecule (toxicity generated after the ADC binds to cell surface antigens in non-tumor tissues and is internalized, also known as "on-target toxicity"), thereby having a higher therapeutic index.

[0347] In some embodiments, the conjugate obtained according to the above method has high stability in the body circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target" toxicity caused by the shedding of toxins in non-target tissues;

[0348] In some embodiments, the bioactive molecule of the conjugate has higher anti-tumor cell activity and thus has an excellent bystander effect. The ADC can more effectively kill tumor cells with high antigen expression and tumor cells with low or no antigen expression in tumor tissues.

[0349] In some embodiments, the antibody-drug conjugates disclosed herein, utilizing the extracellular cleavage ability of their linkers in the tumor microenvironment, can be combined with antibodies that do not have endocytosis ability to form antibody-drug conjugates. Such antibody-drug conjugates still have high anti-tumor activity.

[0350] In some embodiments, the antibody-drug conjugates disclosed herein, utilizing the extracellular cleavage ability and enrichment ability of their linkers in the tumor microenvironment, can be combined with antibodies that lack endocytosis ability and antibodies that lack tumor extracellular antigen binding ability to form antibody-drug conjugates. Such antibody-drug conjugates still have high anti-tumor activity.

[0351] In summary, the ADC disclosed herein has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0352] Figure 1. Cross-detection of humanized antibodies with rat cMet;

[0353] Figure 2. Cross-detection of humanized antibodies with mouse cMet;

[0354] Figure 3. Competition assay between humanized antibody and ABT700 epitope;

[0355] Figure 4. Humanized antibodies block HGF-induced ERK phosphorylation;

[0356] Figure 5. Humanized antibodies induce cMet protein degradation;

[0357] Figure 6. In vivo efficacy of anti-human cMet ADC in the NCI-H358 CDX model (Test 1);

[0358] Figure 7. Changes in mouse body weight during administration of anti-human cMet ADC in the NCI-H358 CDX model (Test 1);

[0359] Figure 8. In vivo efficacy of anti-human cMet ADC in the SW480 CDX model;

[0360] Figure 9. Changes in mouse body weight during administration of anti-human cMet ADC in the SW480 CDX model;

[0361] Figure 10. In vivo efficacy of anti-human c-Met ADC in the NCI-H716 CDX model;

[0362] Figure 11. Changes in mouse body weight during administration of anti-human cMet ADC in the NCI-H716 CDX model;

[0363] Figure 12. Verification of c-Met target expression levels in different tumor cells;

[0364] Figure 13. In vivo efficacy of anti-human cMet ADC in the NCI-H358 CDX model (Test 2);

[0365] Figure 14. Changes in mouse body weight during administration of anti-human cMet ADC in the NCI-H358 CDX model (Test 2);

[0366] Figure 15. In vivo efficacy of anti-human c-Met ADC in the CR5088 PDX model;

[0367] Figure 16. Changes in mouse body weight during administration of anti-human cMet ADC in the CR5088 PDX model;

[0368] Figure 17. ADCC activity test of anti-human cMet antibodies and ADCs on MKN45 cells. The upper figure shows the test curve of the positive control, and the lower figure shows the test curve of the 45A5G10-Hz antibody and its ADC.

[0369] Figure 18. CDC activity test of anti-human cMet antibodies and ADCs on MKN45 cells. The upper figure shows the test curve of the positive control, and the lower figure shows the test curve of the 45A5G10-Hz antibody and its ADC.

[0370] Figure 19. In vivo efficacy of anti-human c-Met ADC in the MKN45 gastric cancer model. DETAILED DESCRIPTION

[0371] The present disclosure is further illustrated below by describing specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure. Reagents or instruments used without indicating the manufacturer are all commercially available conventional products.

[0372] Sequence and its specific information:

[0373] The present disclosure will now be described with reference to the following examples which are intended to illustrate the present disclosure rather than to limit it.

[0374] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present disclosure are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the embodiments describe the present disclosure by way of example and are not intended to limit the scope of protection claimed in the present disclosure.

[0375] The abbreviations in this disclosure have the following meanings. Abbreviations without any meanings have the meanings commonly understood in the art:

[0376] Example 1. Screening and preparation of anti-c-Met antibodies

[0377] 1.1 Antigen information

[0378] 1) Plasmid: Human cMet cDNA ORF Clone was purchased from Sino Biological (Cat. No. HG10463-CH). The nucleotide sequence corresponding to amino acids 25–932 of the extracellular domain of the human cMet protein was then cloned into pTT5-mFc and pTT5-hFc vectors (from Sichuan Sibowo Biotechnology Co., Ltd.), ultimately forming expression plasmids fused with the human IgG Fc region and mouse IgG Fc region, respectively.

[0379] 2) Antigen expression: HEK293E cells (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 using ProA filler (GE, Mabselect XL).

[0380] 3) Commercial antigen proteins: Human cMet extracellular domain recombinant protein (Human HGF R / c-MET Protein, Fc Tag) was from Biopsies (Cat. No. MET-H5256), Human cMet extracellular domain recombinant protein (Human HGF R / c-MET Protein, His Tag) was from Biopsies (Cat. No. MET-H5227), Monkey cMet extracellular domain recombinant protein (c-MET Protein, Cynomolgus, Rhesus, Recombinant (His Tag) was from Sino Biological (Cat. No. 90304-C08H), Rat cMet extracellular domain recombinant protein (Rat-cMet-hFc, c-MET Protein, Rat, Recombinant (hFc Tag) was from Sino Biological (Cat. No. 80004-R02H), Mouse cMet extracellular domain recombinant protein (mouse cMet-ECD-His, c-MET Protein, Mouse, Recombinant (ECD, His Tag) was from Sino Biological (Cat. No. 50622-M08H), and human HGF recombinant protein (HGF Protein, Human, Recombinant) was from Sino Biological (Cat. No. 10463-HNAS).

[0381] 1.2 Immunization and Antibody Screening

[0382] Four female mice each of the CD1 (Weitong Lihua), KM (Weitong Lihua), and Balb / c (Jicui Yaokang) strains were enrolled at approximately 6 weeks of age. Immunizations were performed with cMET-His and hcMET-mFc (Sichuan Sibowo Biotechnology Co., Ltd.). After two booster immunizations, serum titers were measured by ELISA. For mice with high ELISA protein titers, tumor cell MKN-45 affinity titers and MKN45-HGF competition titers were measured by FACS. Finally, spleens and lymph nodes from one CD1, KM, and Balb / c mouse were used to prepare cell suspensions and hybridoma fusions with SP2 / 0 mouse myeloma cells. Initial ELISA screening was performed using the human hMET-ECD-hFc antigen protein, and positive hybridoma clones were selected. Hybridoma cells were cultured in serum-free medium, and the collected monoclonal supernatants were purified using ProA filler to obtain mouse antibodies. The affinity of mouse antibodies to human MET-ECD-hFc and cynomolgus monkey MET-ECD-hFc proteins was tested by ELISA. The affinity of mouse antibodies to MKN45 tumor cells and the blocking effect of MKN45-HGF ligand competition were detected by FACS. After multiple rounds of screening, a total of 18 positive clones were obtained and 18 mouse antibodies were evaluated. The evaluation results of 4 clones with high affinity are shown in Table 1.

[0383] Table 1 Detection of affinity and HGF competitiveness of anti-cMET mouse antibodies

[0384] ND means not detected

[0385] Sequences of four monoclonal clones with high affinity, 55A10G6, 45A5G10, 51D5B2 and 44H10E8, were obtained by fishing. The method for obtaining sequences was as follows: about 1×10 candidate hybridoma cells were collected. 5 RNA was extracted using Trizol, and then reverse transcribed using the PrimeScript RT reagent kit via PolyA to obtain cDNA. Upstream primers were designed upstream of the heavy and light chains, and downstream primers were designed for the heavy chain CH1 region and the light chain CL region, respectively. The product was amplified by PCR and fragments were recovered using an agarose gel extraction kit. Samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The detailed sequences of the mouse antibody variable regions and CDRs are shown in the sequence information table.

[0386] 1.3 Antibody Humanization

[0387] Using the CDR grafting method, the human germline sequence with the highest homology to the original mouse sequence is first found through the conventional BLAST method and used as a template; the CDR of the mouse antibody is transplanted onto the human template to construct a chimera; based on structural analysis of the FR amino acids in the mouse antibody that can retain its original conformation, the corresponding amino acids in the chimera are backmutated to mouse amino acids to maintain the original affinity; the constructed humanized antibody is subjected to calculations and immunogenicity analysis to find highly immunogenic fragments and replace the low-immunogenic fragments. For substitutions of highly immunogenic sites in the CDRs, for example, the 45A5G10 Kabat HCDR2 was replaced from QIRLKSLNYATHYAESVKG (SEQ ID No. 17) to QIRLKSLNYATHYAQSVKG (SEQ ID No. 18), and the 55A10G6 Kabat HCDR2 was replaced from WIFPGSGNTKYIEKFKG (SEQ ID No. 30) to WIFPGSGNTKYSQKFQG (SEQ ID No. 31). Murine antibodies 55A10G6 (abbreviated as 55A10G6) and 45A5G10 (abbreviated as 45A5G10) were humanized to obtain the 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 CDRs are provided according to the IMGT, Chothia and Kabat definitions.

[0388] 1.4 Expression of humanized anti-c-Met antibody

[0389] The heavy chain variable region 55A10G6-hz vh of the humanized antibody was connected 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 connected to the Kappa constant region (SEQ ID NO: 52). The sequence obtained by connection was submitted to Universal Bio for gene synthesis. After codon optimization, it was constructed into the PTT5 vector. After the plasmid was synthesized, HEK293E cells (from Sichuan Sibowo Biotechnology Co., Ltd.) were transfected with PEImax and expressed for about 7 days. The supernatant was collected by centrifugation. The supernatant was purified using ProA filler. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and stored at -20 degrees. Antibody 55A10G6-hz was obtained.

[0390] The heavy chain variable region 45A5G10-hz vh of the humanized antibody was linked 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 linked to the kappa constant region (SEQ ID NO: 52). The resulting sequences were submitted to Universal Bio for gene synthesis. After codon optimization, they were constructed into the pTT5 vector. After plasmid synthesis, HEK293E cells were transfected with PEImax, expressed for approximately 7 days, and the supernatant was collected by centrifugation. The supernatant was purified using ProA filler. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and the cells were stored at -20 degrees Celsius. This yielded the antibody 45A5G10-hz.

[0391] The amino acid sequences of the heavy and light variable regions of a control antibody (ABT700, sequence from KEGG, ID: D11307) were submitted to Universal Biotechnology for gene synthesis. After codon optimization, the construct was constructed into the pTT5 vector. After plasmid synthesis, HEK293E cells were transfected with PEImax. Expression was allowed to continue for approximately 7 days, and the supernatant was collected by centrifugation. The supernatant was then purified using ProA media. The purified antibodies were then ultrafiltered into PBS buffer, the concentration was determined, and the cells were stored at -20°C.

[0392] Example 2: Evaluation of anti-c-Met antibodies

[0393] 2.1 ELISA affinity evaluation of anti-c-Met antibody protein

[0394] The antigen protein cMet-ECD-his was diluted to 1 μg / ml in carbonate buffer (CBS) and then coated with the antigen. The cells were then blocked with 2% BSA (in phosphate buffered saline (PBS)) at 37°C for 2 hours. Serial dilutions of the humanized antibody to be tested were added (starting at 2 μg / mL, 3-fold dilution, 11 concentration points) 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. After quenching with 2 M HCl, the absorbance at 450 nM was read on a microscope. The antigen protein anti-His-Rabbit Fc (from Chengdu Apak Biotechnology Co., Ltd.) was diluted to 1 μg / ml in CBS and then coated with the antigen. The cells were then blocked with 2% BSA (in PBS) at 37°C for 2 hours. 0.5 μg / ml cyno.cMet-ECD-His was added. Incubate at 37°C for 2 hours, then add serially diluted humanized antibodies to be tested (starting at 2 μg / mL, 3-fold dilution, 11 concentration points) within 2 hours, 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, terminate with 2 M HCl, and read the absorbance at 450 nM on a spectrometer.

[0395] The experimental results are shown in Table 2. The two humanized antibodies have high affinities for both human and cynomolgus monkey Met proteins, and have significantly stronger affinity for cynomolgus monkey Met than ABT700.

[0396] Table 2. ELISA affinity evaluation of humanized antibodies

[0397] 2.3 Flow cytometry affinity evaluation of anti-c-Met antibodies

[0398] MKN45 cells (Nanjing Kebai, CBP60488) were collected by trypsin digestion and centrifugation, washed three times with pre-cooled PBS, and resuspended with 1% BSA (in PBS) at 2^10 per well. 5 Cells (50 μl) were plated into 96-well conical bottom plates. The anti-cMet humanized antibody to be tested was serially diluted with 1% BSA, starting at 20 μg / ml, with a 4-fold dilution gradient and 8 dilution points. 50 μl of the diluted antibody was mixed with the cells in the conical bottom 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, Cat. No. 410712) was added to each well and incubated at 4°C for 0.5 hour. After washing three times with pre-chilled PBS, the cells were resuspended and detected by flow cytometry (Beckman, Cytoflex).

[0399] The experimental results are shown in Table 3. Both humanized antibodies have high affinity for MKN45 cells, and the affinity of both antibodies for MKN45 is higher than that of the control antibody ABT700.

[0400] Table 3. Flow cytometry affinity evaluation of humanized antibodies

[0401] 2.4 Dynamic affinity evaluation of anti-c-Met antibodies

[0402] The dynamic affinity of humanized antibodies was determined using ForteBio. The experimental steps are as follows: 1. Sensor preparation: Remove the ProA sensor and pre-wet it with PBST (pH 7.4) for 10 minutes. 2. Sample dilution: Dilute the antibodies to be immobilized to 5 μg / ml. For the antigen h.cMet-ECD-His (Bipsys, Catalog No. MET-H5227), start at 500 nM and dilute it 2-fold over five concentration points, including a zero concentration point. 3. Set up the program: Place the sensor plate and sample plate, start the program, and regenerate the sensor with 20 mM glycine (pH 1.7). 4. Analyze the data using Octet analysis software. The results are shown in Table 4. Both antibodies 45A5G10-Hz and 55A10G6-Hz exhibit high dynamic affinity for human cMet protein.

[0403] Table 4. Dynamic affinity testing of humanized antibodies

[0404] 2.5 Quality Assay of Anti-c-Met Antibodies

[0405] 1) The purity of the humanized antibody was determined by SEC using the following assay method:

[0406] Instrument: Waters Alliance e2695 HPLC;

[0407] Chromatographic column: Thermo MabPac SEC-1, 5 μm, 7.8*300 mm;

[0408] Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA;

[0409] Instrument parameters: sample chamber temperature: 8°C; column temperature: 30°C; flow rate: 0.5 ml / min; injection volume: 20 μg; detection wavelength: 280 nm; isocratic operation: 30 min.

[0410] 2) The hydrophilicity of the humanized antibody was tested by HIC. The test method was as follows: Hydrophilicity was tested using a Tosoh hydrophobic chromatography column (TOSOH Tskgel Buty-NPR (2.5), 4.6*100) on an Agilent HPLC instrument. Mobile phase A consisted of 1.5 M (NH4)2SO4 and mobile phase B consisted of 25 mM Na2HPO4 (pH = 7.0) + 25% IPA. Instrument parameters were set as sample chamber temperature: 8°C, column temperature: 30°C, flow rate: 0.5 mL / min, and detection wavelength: 280 nm. The humanized antibody sample to be tested was diluted with mobile phase A to a final concentration of 1 mg / mL, and 20 μL of the sample was injected for gradient elution.

[0411] 3) Determine the Tm value of the humanized antibody using DSF to reflect the thermal stability of the antibody. The experimental steps are as follows: dilute the humanized antibody sample to be tested to 1 mg / mL with PBS; dilute the dye SYPRO Orange dye (Thermo #56651) to 40X with ddH2O; the reaction system is: 12.5uL sample + 2.5uL 40X dye + 5uL ddH2O; seal the membrane and centrifuge briefly; detect by Q-PCR, Q-PCR parameters set as follows: Target (ROX), program (25°C, 3 min; 1% rate, 95°C; 95°C, 2 min).

[0412] The results of SEC, HIC, and Tm values ​​are shown in Table 5. SEC results indicated that both humanized antibodies had high purity, >90%. HIC results indicated that both humanized antibodies bound weakly to the hydrophobic column, with short retention times and good hydrophilicity. Tm values ​​indicated that both humanized antibodies possessed good thermal stability.

[0413] Table 5. Quality evaluation of humanized antibodies

[0414] 2.6 Cross-detection of anti-c-Met antibodies with rat and mouse cMet

[0415] The antigen protein Rat-cMet-hFc was diluted to 0.5 μg / ml in CBS and then coated with the antigen. The cells were then blocked with 2% BSA (in PBS) at 37°C for 2 hours. A serial dilution of the humanized antibody to be tested (biotin-labeled) was added (starting at 2 μg / mL, 3-fold dilution, 11 concentration points) and incubated at 37°C for 2 hours. An 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. After quenching with 2 M HCl, the absorbance was read at 450 nM.

[0416] The protein anti-His-hFc (from Chengdu Apak Biotechnology Co., Ltd.) was diluted in CBS at 0.5 μg / ml and then coated. The cells were then blocked with 2% BSA (in PBS) at 37°C for 2 hours, and 1 μg / ml mouse cMet-ECD-His was added and incubated at 37°C for 2 hours. After 2 hours, a serial dilution of the humanized antibody to be tested (starting at 2 μg / mL, 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 absorbance value at 450 nM was read after termination with 2 M HCl.

[0417] The experimental results are shown in Figures 1 and 2. The two humanized antibodies, 45A5G10-Hz and 55A10G6-Hz, have no cross-reactivity with rat and mouse cMet (i.e., they do not bind to rat and mouse cMet).

[0418] 2.7 Evaluation of anti-c-Met antibody endocytic activity

[0419] MKN45 cells were trypsinized and collected by centrifugation. They were incubated with 10 μg / mL of the humanized antibody to be tested at 4°C for 1 hour, washed three times with PBS, and resuspended in DMEM + 10% FBS, pre-warmed at 37°C. The cells were divided into three aliquots and incubated at 37°C for 0, 2, and 4 hours, respectively. After washing three times with pre-chilled PBS, 1.2 μL of anti-human APC fluorescent secondary antibody was added and incubated at 4°C for 0.5 hours. The cells were washed three times with PBS and resuspended for analysis. The endocytosis rate was calculated according to the following formula: endocytosis rate (%) = [1 - (average fluorescence value of the test sample at that time point - average fluorescence value of the negative control sample at that time point) / (average fluorescence value of the test sample at 0 hour - average fluorescence value of the negative control sample at 0 hour)] * 100.

[0420] The experimental results are shown in Table 6. The endocytic activity of antibodies 45A5G10-Hz and 55A10G6-Hz was higher than that of ABT700 at the same time, and the endocytic rate could reach more than 60% in 4 hours.

[0421] Table 6. Humanized antibody endocytosis activity detection

[0422] 2.8 Evaluation of anti-c-Met antibody epitope competition

[0423] The cells were coated with CBS-coated anti-His-Rabbit Fc protein at 0.5 μg / ml and blocked with 2% BSA (in PBS) at 37°C for 2 hours. 1 μg / ml h.cMet-ECD-His was added and incubated at 37°C for 2 hours. After 2 hours, a gradient dilution of the humanized antibody to be tested (starting at 10 μg / mL, 3-fold dilution, 11 concentration points) was added to a final concentration of 5 μg / ml. The cells were incubated at room temperature for 0.5 hours. After the incubation, 200 ng / ml ABT700-Biotin was added to a final concentration of 100 ng / ml. The cells were 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. The cells were terminated with 2 M HCl and the absorbance at 450 nM was read on a spectrometer.

[0424] The experimental results are shown in Figure 3. The two humanized antibodies, 45A5G10-Hz and 55A10G6-Hz, do not compete with ABT700 for epitopes.

[0425] 2.9 Competition evaluation of anti-c-Met antibodies with HGF ligands

[0426] cMET is a tyrosine kinase receptor expressed on the cell membrane. It binds to its ligand HGF through the Sema domain, thereby inducing a downstream phosphorylation cascade reaction, ultimately promoting cell proliferation. Therefore, the competitive effect of humanized antibodies on ligand binding and their inhibition of HGF-induced ERK phosphorylation were tested.

[0427] MKN45 cells were collected by trypsin digestion and centrifugation, washed three times with pre-cooled PBS, and resuspended in 1% BSA (in PBS) at 2^10 per well. 5 100 μl of cells (50 μl) were plated into 96-well conical-bottom plates. Humanized anti-cMet antibody was serially diluted with 1% BSA, starting at 15 μg / ml and ending at 5 μg / ml, with a 3-fold dilution gradient and 8 dilution points. 50 μl of the diluted antibody was mixed with the cells in the conical-bottom plate. HGF-His-biotin (from Chengdu Apak Biotechnology Co., Ltd.) was diluted to 150 ng / ml with 1% BSA, and 50 μl / well was added to the plate to a final ligand concentration of 50 ng / ml. After thorough mixing, the cells were 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-biotin PE fluorescent secondary antibody, BioLegend, Cat. No. 405204) was added to each well and incubated at 4°C for 0.5 hour. After washing three times with pre-chilled PBS, the cells were resuspended and analyzed by flow cytometry (Beckman, Cytoflex).

[0428] The experimental results are shown in Table 7. Both humanized antibodies have competitive activity with HGF ligand on MKN45 cells, and the competitive activity is higher than that of ABT700.

[0429] Table 7 Competition assay between humanized antibodies and HGF ligands

[0430] 2.10 Anti-c-Met antibody inhibits HGF-induced ERK phosphorylation

[0431] MDA-MB-468 cells (ATCC, Catalog No. HTB-132) were trypsinized and counted, and the cells were resuspended in L15 medium to 2*10^5 / ml. The cells were then plated into 24-well plates with 1 ml of cell suspension per well, for a total of 4 wells. The cells were then returned to 37°C cell culture medium for overnight incubation. After 16 hours, the cells in 6 wells were divided into groups: untreated, HGF (Sino Biological, Catalog No.: 10463-HNAS), HGF + isotype control (hIgG1, manufactured by Suzhou Yilian Biopharmaceutical Co., Ltd.), HGF + 45A5G10-Hz, and HGF + 55A10G6-Hz. The corresponding sterile samples were then added to the corresponding cell wells. The test antibody was added separately 15 minutes in advance (final concentration 15ug / ml). 15 minutes later, HGF was added to a final concentration of 100ng / ml. The cells were returned to 37°C cell culture medium for incubation for 15 minutes. After 15 minutes, cells were rinsed three times with PBS and SDS lysis buffer was added directly to each well to lyse the cells and harvest proteins. Harvested proteins were analyzed by conventional SDS-PAGE and Western blot. The detection antibody for phosphorylated ERK was from CST (Cat. #5726S), and the detection antibody for GAPDH was from Sino Biological (Cat. No. 100242-MM05). Both primary antibodies were diluted at 1:2000 and incubated overnight at 4°C.

[0432] The results are shown in Figure 4. HGF treatment alone can significantly induce ERK phosphorylation in MDA-MB-468 cells, while the advance addition of antibodies 45A5G10-Hz and 55A10G6-Hz can completely block HGF-induced ERK phosphorylation.

[0433] 2.11 Humanized Antibody Induces cMet Protein Degradation

[0434] MKN-45 cells were trypsinized and counted, and the cells were resuspended to 2*10^5 / ml in RPMI 1640 + 10% FBS (fetal bovine serum) + Ps (penicillin-streptomycin). The cells were then plated into 24-well plates with 1 ml of cell suspension per well, for a total of 4 wells. The cells were then returned to 37°C cell culture medium and incubated overnight. After 16 hours, the cells in the four wells were grouped into groups: untreated, isotype control (hIgG1), 45A5G10-Hz, and 55A10G6-Hz. The corresponding sterile samples were then added to the corresponding wells at a final antibody concentration of 20 μg / ml. The cells were returned to 37°C cell culture medium and incubated for 60 hours. After 60 hours, the cells were rinsed three times with PBS, and SDS lysis buffer was added directly to each well to lyse the cells and harvest the protein. The harvested proteins were subjected to conventional SDS-PAGE and Western blot analysis. The cMet detection antibody was from ProteinTech (Cat. No. 25869-1-AP), and the GAPDH detection antibody was from Sino Biological (Cat. No. 100242-MM05). The dilution ratio of both primary antibodies was 1:2000, and the cells were incubated at 4°C overnight.

[0435] As shown in Figure 5, compared with the isotype control antibody IgG1 treatment, the anti-cMet antibodies 45A5G10-Hz and 55A10G6-Hz can significantly induce cMet degradation.

[0436] Example 3. Preparation of anti-c-Met ADC

[0437] Example 3.1 Preparation of anti-c-MET-B81 ADC

[0438] Wherein Ab is a c-MET antibody (such as 45A5G10-Hz and 55A10G6-Hz described below), and q can be determined by the data provided below.

[0439] 3.1.1 45A5G10-HZ-B81 (DAR8) Sample Preparation

[0440] A total of 20 mg of the anti-c-Met antibody 45A5G10-HZ was added to a sodium edetate solution with a final concentration of 1 mM and mixed. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution; a 20 mmol / L TCEP solution with a 6.5-fold molar equivalent of the antibody was added, mixed, and allowed to stand at 37 ° C for 90 minutes; 10 mmol / L B81 dissolved in dimethyl sulfoxide (prepared with reference to DL-037 in Example 2.37 of WO2022170971) with a 15-fold molar equivalent of the antibody was added to the above solution system, mixed, and allowed to stand at 37 ° C for 3 hours to obtain a coupled sample. After the reaction, the sample was replaced with a 20 mM histidine buffer with a pH of 6.0 using a 30 KDa ultrafiltration tube and low molecular weight substances were removed. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC 45A5G10-HZ-B81 (DAR8).

[0441] RP-LC / MS was used to determine the DAR value of the coupled sample.

[0442] LC / MS Model:

[0443] Liquid phase parameters

[0444] The results showed that the light chain of the 45A5G10-HZ-B81 (DAR8) sample was conjugated to 0-1 toxin molecules (LC and DAR1 ratios were 0% and 100.0%, respectively), and the heavy chain was conjugated to 0-3 toxin molecules (mAb, DAR1, DAR2, and DAR3 ratios were 0%, 0%, 0%, and 100.0%, respectively). Based on this, the conjugation ratio (DAR value) of the 45A5G10-HZ-B81 (DAR8) sample was calculated to be 8.0. It can be inferred that q is 8.

[0445] In the above, mAb represents an unconjugated monoclonal antibody; LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate comprising a light chain or heavy chain conjugated to one toxin molecule; DAR2 represents a conjugate comprising a light chain or heavy chain conjugated to two toxin molecules; and DAR3 represents a conjugate comprising a light chain or heavy chain conjugated to three toxin molecules. The theoretical molecular weight of a monoclonal antibody is calculated based on the G0F glycoform. This applies to mAb, LC, HC, DAR1, DAR2, and DAR3 below.

[0446] 3.1.2 45A5G10-HZ-B81 (DAR4) Sample Preparation

[0447] A total of 20 mg of the anti-c-Met antibody 45A5G10-HZ was added to a sodium edetate solution with a final concentration of 1 mM and mixed. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution; a 20 mmol / L TCEP solution with a 2.7-fold molar equivalent of the antibody was added, mixed, and allowed to stand at 5°C for 5 h; 10 mmol / L B81 dissolved in dimethyl sulfoxide (prepared with reference to DL-037 in Example 2.37 of WO2022170971) with a 6.5-fold molar equivalent of the antibody was added to the above solution system, mixed, and allowed to stand at room temperature for 1 hour to obtain a coupled sample. After the reaction, the sample was replaced with a 20 mM histidine buffer with a pH of 6.0 using a 30 KDa ultrafiltration tube and low molecular weight substances were removed. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC45A5G10-HZ-B81 (DAR4).

[0448] Determination of DAR value of coupled samples using Native-LC / MS

[0449] LC / MS Model:

[0450] Liquid phase parameters

[0451] The results showed that the entire antibody of the 45A5G10-HZ-B81 (DAR4) sample was coupled to 0 to 8 toxin molecules (the proportions of mAb, q is 2, q is 4, q is 6, and q is 8 were 0%, 22%, 56%, 23%, and 0%, respectively). Based on this, the coupling ratio (DAR value) of the 45A5G10-HZ-B81 (DAR4) sample was calculated to be 4.0.

[0452] 3.1.3: Preparation of 55A10G6-HZ-B81 (DAR8)

[0453] A total of 20 mg of the anti-c-Met antibody 55A10G6-HZ was added to a sodium edetate solution with a final concentration of 1 mM and mixed. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution; a 20 mmol / L TCEP solution (6.5 times the molar equivalent of the antibody) was added, mixed, and allowed to stand at 37°C for 90 minutes; B81 (10 mmol / L) dissolved in dimethyl sulfoxide (DMSO) was added to the above solution system, mixed, and allowed to stand at 37°C for 3 hours to obtain a coupled sample. After the reaction, the sample was replaced with a 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 55A10G6-HZ-B81 (DAR8).

[0454] Referring to the DAR values ​​of the conjugated samples determined by LC / MS in 3.1.1 above, the light chain of the 55A10G6-HZ-B81 sample was conjugated to 0-1 toxin molecules (LC, DAR1 ratios of 0% and 100.0%, respectively), and the heavy chain was conjugated to 0-3 toxin molecules (mAb, DAR1, DAR2, and DAR3 ratios of 0%, 0%, 0%, and 100.0%, respectively). Based on this, the conjugation ratio (DAR value) of the 55A10G6-HZ-B81 sample was calculated to be 8.0. It can be inferred that q is 8.

[0455] Example 3.2 Preparation of anti-c-MET-vc-MMAE

[0456] Wherein Ab is a c-MET antibody (such as ABT700 described below), and q can be determined based on the data provided below.

[0457] 3.2.1 ABT700-vc-MMAE (DAR9.7) sample preparation

[0458] A total of 20 mg of the anti-c-Met antibody ABT700 was added to a sodium edetate solution with a final concentration of 1 mM and mixed. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution; a 20 mmol / L TCEP solution with a molar equivalent of 6.5 times the antibody was added, mixed, and allowed to stand at 37°C for 90 minutes; to the above solution system, 10 mmol / L vc-MMAE (CAS No.: 646502-53-6, purchased from MedChemExpress) dissolved in dimethyl sulfoxide with a molar equivalent of 15 times the antibody was added, mixed, and allowed to stand at 37°C for 3 hours to obtain a coupled sample. After the reaction, the sample was replaced with a 20 mM histidine buffer with a pH of 6.0 using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC ABT700-vc-MMAE (DAR9.7).

[0459] Referring to the DAR value of the coupled sample by LC / MS in 3.1.1 above, the light chain of the ABT700-Vc-MMAE (DAR9.7) sample was coupled to 1 to 3 toxin molecules (the ratios of DAR1, DAR2, and DAR3 were 64.3%, 29.8%, and 5.9%, respectively), and the heavy chain was coupled to 3 to 4 toxin molecules (the ratios of DAR3 and DAR4 were 56.4% and 43.6%, respectively). Based on this, the coupling ratio (DAR value) of the ABT700-Vc-MMAE (DAR9.7) sample was calculated to be 9.7.

[0460] 3.2.2 ABT700-vc-MMAE (DAR4) sample preparation

[0461] A total of 20 mg of the anti-c-Met antibody ABT700 was added to a sodium edetate solution with a final concentration of 1 mM and mixed. The pH of the sample was adjusted to 7.5 with a 0.5 M disodium hydrogen phosphate solution. A 20 mmol / L TCEP solution (2.8 times the molar equivalent of the antibody) was added, mixed, and allowed to stand at room temperature for 90 minutes. To the above solution system, 10 mmol / L vc-MMAE (CAS No.: 646502-53-6, purchased from MedChemExpress) dissolved in dimethyl sulfoxide (4.2 times the molar equivalent of the antibody) was added, mixed, and allowed to stand at room temperature for 3 hours to obtain a coupled sample. After the reaction, the sample was replaced with a 20 mM histidine buffer with a pH of 6.0 using a 30 kDa ultrafiltration tube to remove low molecular weight substances. Finally, the sample was concentrated to obtain a solution containing the anti-c-Met antibody ADC ABT700-Vc-MMAE (DAR4).

[0462] Referring to the DAR value of the coupled sample determined by LC / MS in 3.1.1 above, the light chain of the ABT700-VC-MMAE (DAR4) sample was coupled to 0 to 1 toxin molecules (the ratios of LC and DAR1 were 52.2% and 47.8%, respectively), and the heavy chain was coupled to 0 to 3 toxin molecules (the ratios of mAb, DAR1, DAR2, and DAR3 were 17.5%, 47.2%, 22.9%, and 12.5%, respectively). Based on this, the coupling ratio (DAR value) of the ABT700-VC-MMAE (DAR4) sample was calculated to be 4.0.

[0463] Example 4. In vitro evaluation of anti-human c-Met antibodies and ADCs

[0464] 4.1 Anti-human cMet ADC protein affinity detection

[0465] Anti-His-RabbitFc was coated with CBS protein at 0.5 μg / ml, and blocked with 2% BSA (in PBS) at 37°C for 2 hours. 1 μg / ml h.cMet-ECD-His was added and incubated at 37°C for 2 hours. After 2 hours, a serial dilution of the antibody or ADC to be detected (starting at 2 μg / mL, 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 color was terminated with 2 M HCl and the absorbance value at 450 nM was read on a microscope.

[0466] 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 of ABT700 decreased significantly after being coupled to ADC.

[0467] Table 8 Protein affinity detection before and after cMET antibody conjugation into ADC

[0468] 4.2 Anti-human cMet ADC cell affinity detection

[0469] Test 1

[0470] NCI-H358 (human non-small cell lung cancer cells, purchased from ATCC, catalog number CRL-5807), LS1034 cells (human colon adenocarcinoma cells, Nanjing Kebai, catalog number CBP60013), NCI-H69 (human small cell lung cancer cells, Wuhan Punuosai, catalog number CL-0677) and NCI-H716 (human colorectal adenocarcinoma cells, purchased from ATCC, catalog number CCL-251) cells were harvested 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 Cells (50ul) were plated into 96-well conical bottom plates. The anti-cMet humanized antibody ADC was serially diluted with 1% BSA, starting at 15μg / ml, with a 4-fold dilution gradient and 8 dilution points. 50ul of the diluted ADC was mixed with the cells in the conical bottom plate and incubated at 4°C for 1 hour. After washing three times with pre-chilled PBS, 100ul of 1% BSA (containing 1uL of anti-human APC fluorescent secondary antibody, BioLegend, Cat. No. 410712) was added to each well and incubated at 4°C for 0.5 hour. After washing three times with pre-chilled PBS, the cells were resuspended and detected by flow cytometry (Beckman, Cytoflex).

[0471] The experimental results are shown in Table 9. The ADCs of the two humanized antibodies conjugated to B81 exhibited high affinity for NCI-H358, LS1034, NCI-H69, and NCI-H716 cells. The affinities of the ADCs for both antibodies were higher than those of ABT700-VcMMAE (DAR 9.7) on these three tumor cell lines. The ADCs for both antibodies also exhibited higher affinities for NCI-H716 cells than ABT700-VcMMAE (DAR 4).

[0472] Table 9. Anti-human cMet ADC cell affinity detection

[0473] Test 2

[0474] MKN45 cells (derived from JCBR JCRB0254) were collected by trypsin digestion and centrifugation, washed three times with pre-cooled PBS, and diluted to 1×10 6 cells / mL, 1×10 per well 5 Cells (100 μL) were plated into 96-well conical bottom plates, and 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ were serially diluted with FACS buffer, starting at 25 nM, with a 4-fold dilution gradient and 8 dilution points. The cells were mixed with the cells in the conical bottom plate at 100 μL / well, incubated at 4°C for 0.5 h, washed three times with pre-cooled PBS, and 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, washed three times with pre-cooled PBS, and then resuspended for flow cytometry detection.

[0475] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had high binding activity to MKN45, and the affinity of 45A5G10-HZ and 45A5G10-HZ-B81(DAR8) on MKN45 was similar. 50 They are 0.215nM and 0.189nM respectively.

[0476] 4.3 ADC in vitro killing activity detection

[0477] Test 1

[0478] Tumor cells with good growth status were digested and centrifuged to collect SW480 cells (purchased from ATCC, catalog number CCL-228), NCI-H358 cells (purchased from ATCC, catalog number CRL-5807), LS1034 cells (Nanjing Kebai, catalog number CBP60013), and NCI-H716 cells (purchased from ATCC, catalog number CCL-251). The cells were directly collected by centrifugation. Resuspend cells in 1640+5% FBS+Ps, count, and plate 5000 (LS1034 and NCI-H716) or 3000 (SW480 and NCI-H358) cells per well in a volume of 100 μL. Dilute the ADC to be tested in the corresponding resuspension culture medium of the above three cell types, starting at 300 μg / mL (2 μM), and dilute 3-fold to 11 concentration points. Add 100 μL / well to the plate to a final ADC concentration of 150 μg / mL. Incubate at 37°C for 5 days (LS1034, NCI-H358) or 7 days (NCI-H716, SW480). After incubation, add CCK8 at 20 μL / well and react for 1-3 hours. Read the results at 450 nm on a microplate reader and import them into Graphpad Prism for curve fitting.

[0479] The experimental results are shown in Table 10. Both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) can effectively kill four tumor cells: LS1034, NCI-H358, NCI-H716, and SW480. In LS1034 cells, the cell killing activity of 45A5G10-Hz-B81 (DAR8) is comparable to that of ABT700-VcMMAE (DAR9.7), while 55A10G6-Hz-B81 (DAR8) is slightly weaker than that of ABT700-VcMMAE (DAR9.7); in NCI-H358 cells, 45A5G10-Hz-B81 (DAR8) has stronger killing activity than 55A10G6-Hz-B81 (DAR8); in NCI-H716, 45A5G10-Hz-B81 (DAR8) has stronger killing activity than 55A10G6-Hz-B81 (DAR8); in NCI-H358 cells, 45A5G10-Hz-B81 (DAR8) has stronger killing activity than 55A10G6-Hz-B81 (DAR8). 6 cells, the killing activity of 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) was stronger than that of ABT700-VcMMAE (DAR4); in SW480 cells, the cell killing activity of 45A5G10-Hz-B81 (DAR8) was stronger than that of ABT700-VcMMAE (DAR4), while that of 55A10G6-Hz-B81 (DAR8) was equivalent to that of ABT700-VcMMAE (DAR4).

[0480] Table 10. In vitro cell killing activity assay of anti-human cMet ADC

[0481] Test 2

[0482] 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, and the cells were cultured in RPMI containing 10% FBS. Resuspend the cells in 1640 medium, count them, and plate 1500 (NCI-H441 / NCI-H358 / MKN45) cells per well in a volume of 135 μL. Dilute the 45A5G10-HZ-B81 (DAR8) to be tested with the corresponding resuspension medium of the above three cell types, starting at 20,000 nM, 4-fold dilution, 9 concentration points, further dilute with culture medium to a starting concentration of 500 nM, and add 15 μL / well to the plate to a final ADC concentration of 50 nM. Incubate at 37°C for 6 days. After incubation, add CellTiter-Glo working solution at 75 μL / well, shake on an orbital shaker for 2 minutes to induce cell lysis, and place at room temperature for 10 minutes to stabilize the luminescent signal. Detect the luminescent signal with a microplate reader.

[0483] The experimental results are shown in Table 11. The results show that 45A5G10-HZ-B81 (DAR8) has a strong anti-proliferative effect on NCI-H441 cells, with an IC50 of 0.031 nM. 45A5G10-HZ-B81 (DAR8) has an anti-proliferative effect on NCI-H358 cells, with an IC50 of 0.161 nM. 45A5G10-HZ-B81 (DAR8) has a strong anti-proliferative effect on MKN45 cells, with an IC50 of 0.057 nM.

[0484] Table 11. Anti-proliferative effects of anti-human cMet ADC

[0485] 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. While NCI-H358 cells showed significantly lower target expression than NCI-H441 cells (FACS verification of c-Met target expression is shown in Figure 12), it still exhibited a maximum inhibition rate of >40%.

[0486] 4.4 Forte Bio Detects the Affinity of Anti-c-Met Antibodies and ADCs to Human Fc Receptors and Complement C1q

[0487] 4.4.1: Affinity detection of FcγRI with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0488] The Fc receptor FcγRI, also known as CD64, binds to the Fc terminus of IgG antibodies and participates in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to Fc receptors affects the safety and efficacy of the antibody.

[0489] In this experiment, the Fortebio Octet molecular interaction instrument was used to detect the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ with FcγRI to evaluate their potential ADCC and ADCP activities.

[0490] The experimental method for measuring the affinity constants of the corresponding antibodies for FcγRI using the Fortebio Octet molecular interaction instrument is briefly described as follows: Sample dilution buffer consists of PBS containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 5 μg / mL solution of FcγRI (purchased from ACRO Biosystems, Cat. No. FCA-H52H1) was added to the HIS1K sensor to immobilize FcγRI on the sensor surface. Binding and dissociation parameters of the antibodies to FcγRI were measured in buffer at antibody concentrations of 200, 100, 50, 25, 12.5, and 6.25 nM. After the antigen-immobilized sensor equilibrated in buffer for 60 seconds, binding of the sensor-immobilized FcγRI to each antibody was measured for 60 seconds; dissociation of FcγRI from the antibody was measured for 120 seconds. Data were analyzed using DataAnalysis11 to determine the affinity constants for each antibody and FcγRI.

[0491] The results of affinity constant determinations of FcγRI with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 12 below.

[0492] Table 12. Kinetic parameters for binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRI

[0493] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRI.

[0494] 4.4.2: Affinity detection of FcγRIIIa_V176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0495] Fc receptor FcγRIIIa_V176, also known as (CD16a_V176), can bind to the Fc end of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0496] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIIa_V176 were detected using the Fortebio Octet molecular interaction instrument to evaluate their potential ADCC activity.

[0497] The affinity constants of the corresponding antibodies for FcγRIIIa_V176 were determined using the Fortebio Octet molecular interaction instrument. The experimental method is briefly described as follows: Sample dilution buffer was PBS 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 No. CD8-H52H4) was added to the HIS1K sensor to immobilize FcγRIIIa_V176 on the sensor surface. Binding and dissociation parameters of the antibodies to FcγRIIIa_V176 were determined in buffer at antibody concentrations of 2500, 1250, 625, 312.5, 156.25, and 78.125 nM. After the antigen-immobilized sensor was equilibrated in buffer for 60 seconds, the binding of FcγRIIIa_V176 immobilized on the sensor to each antibody was measured for 60 seconds. The dissociation of FcγRIIIa_V176 from the antibody was measured for 60 seconds. The data was analyzed using Data Analysis 11 to determine the affinity constants for each antibody and FcγRIIIa_V176.

[0498] The results of affinity constant determination for FcγRIIIa_V176, 45A5G10-HZ-B81 (DAR8), and 45A5G10-HZ are shown in Table 13 below.

[0499] Table 13. Kinetic parameters of binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIIa_V176

[0500] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIIa_V176.

[0501] 4.4.3: Affinity detection of FcγRIIIa_F176 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0502] Fc receptor FcγRIIIa_F176, also known as (CD16a_F176), can bind to the Fc end of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0503] The binding experiment of the two samples with FcγRIIIa_F176 (purchased from ACRO Biosystems, product number CDA-H 522 0) was consistent with the CD16a (V 176) binding experiment except that the analyte concentration range in the binding step was optimized to (5000, 2500, 1250, 625, 312.5, 156.25 nM).

[0504] The results of affinity constant determination for FcγRIIIa_F176, 45A5G10-HZ-B81 (DAR8), and 45A5G10-HZ are shown in Table 14 below.

[0505] Table 14: Kinetic parameters for binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIIa_F176

[0506] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIIa_F176.

[0507] 4.4.4: Affinity detection of FcγRIIa_H167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0508] Fc receptor FcγRIIa_H167, also known as (CD32a_H167), can bind to the Fc end of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0509] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIa_H167 were detected using the Fortebio Octet molecular interaction instrument to evaluate their potential ADCC activity.

[0510] The affinity constants of the corresponding antibodies for FcγRIIa_H167 were determined using the Fortebio Octet molecular interaction instrument. The experimental method is briefly described as follows: Sample dilution buffer was PBS containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 10 μg / mL solution of FcγRIIa_H167 (purchased from ACRO Biosystems, Cat. No. CD1 H5223) was added to the HIS1K sensor to immobilize FcγRIIa_H167 on the sensor surface. Binding and dissociation parameters of the antibodies to FcγRIIa_H167 were determined in buffer at antibody concentrations of 5000, 2500, 1250, 625, 312.5, and 156.25 nM. After the antigen-immobilized sensor was equilibrated in buffer for 60 seconds, the binding of FcγRIIa_H167 immobilized on the sensor to each antibody was measured for 60 seconds. The dissociation of FcγRIIa_H167 from the antibody was measured for 60 seconds. The data was analyzed using Data Analysis 11 to determine the affinity constants for each antibody and FcγRIIa_H167.

[0511] The results of affinity constant determination for FcγRIIa_H167, 45A5G10-HZ-B81 (DAR8), and 45A5G10-HZ are shown in Table 15 below.

[0512] Table 15. Kinetic parameters for binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIa_H167

[0513] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIa_H167.

[0514] 4.4.5: Affinity detection of FcγRIIa_R167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0515] Fc receptor FcγRIIa_R167, also known as (CD32a_R167), can bind to the Fc end of IgG antibodies and participate in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0516] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIa_R167 were detected using the Fortebio Octet molecular interaction instrument to evaluate their potential ADCC activity.

[0517] The binding experiment of the two samples with FcγRIIa_R167 (purchased from ACRO Biosystems, catalog number CDA-H 522 1) was the same as that of the FcγRIIa_H167 binding experiment except that the binding steps were the same.

[0518] The results of affinity constant determination for FcγRIIa_R167 with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 16 below.

[0519] Table 16. Kinetic parameters for binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIa_R167

[0520] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIa_R167.

[0521] 4.4.6: Affinity testing of FcγRIIb / c with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0522] Fc receptor FcγRIIb / c, also known as (CD32b / c), can bind to the Fc end of IgG antibodies, negatively regulate the function of immune cells, inhibit the activation and proliferation of immune cells, and inhibit the secretion of cytokines.

[0523] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIb / c were detected using the Fortebio Octet molecular interaction instrument to evaluate the binding ability of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIb / c.

[0524] The affinity constants of the corresponding antibodies for FcγRIIb / c were determined using the Fortebio Octet molecular interaction instrument. The experimental method is briefly described as follows: Sample dilution buffer consists of PBS 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 No. CDB-H5228) was added to the HIS1K sensor to immobilize FcγRIIb / c on the sensor surface. Binding and dissociation parameters of the antibodies to FcγRIIb / c were measured in buffer at antibody concentrations of 10,000, 5,000, 2,500, 1,250, 625, and 312.5 nM. After the antigen-immobilized sensor equilibrated in buffer for 60 seconds, binding of the sensor-immobilized FcγRIIb / c to each antibody was measured for 60 seconds; dissociation of FcγRIIb / c from the antibody was measured for 60 seconds. The data were analyzed using Data Analysis 11 to obtain the affinity constants of each antibody and FcγRIIb / c.

[0525] The affinity constants of FcγRIIb / c for 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 17 below.

[0526] Table 17. Kinetic parameters for binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcγRIIb / c

[0527] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcγRIIb / c.

[0528] 4.4.7: Affinity detection of FcRn with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0529] The Fc receptor FcRn can bind to the Fc end of the IgG antibody, protecting the antibody macromolecules from destruction, and then release the antibody macromolecules in a blood environment with a pH of 7.4.

[0530] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcRn were detected using the Fortebio Octet molecular interaction instrument to evaluate the binding ability of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcRn.

[0531] The experimental method for measuring the affinity constants of the corresponding antibodies for FcRn using the Fortebio Octet molecular interaction instrument is briefly described as follows: Sample dilution buffer consists of PBS containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). A 2 μg / mL solution of FcRn (purchased from ACRO) was added to the HIS1K sensor to immobilize FcRn on the sensor surface. Binding and dissociation parameters of the antibodies to FcRn were measured in buffer at antibody concentrations of 1000, 500, 250, 125, 62.5, and 31.25 nM. After the antigen-immobilized sensor equilibrated in buffer for 60 seconds, binding of the sensor-immobilized FcRn to each antibody was measured for 60 seconds; dissociation of the FcRn-antibody pair was measured for 60 seconds. Data were analyzed using DataAnalysis11 to determine the affinity constants for each antibody and FcRn.

[0532] The results of the affinity constant determinations for FcRn, 45A5G10-HZ-B81 (DAR8), and 45A5G10-HZ are shown in Table 18 below.

[0533] Table 18 Kinetic parameters of binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to FcRn

[0534] The results showed that 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ had similar binding activities to FcRn.

[0535] 4.4.8: Determination of affinity of C1q to 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ

[0536] 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.

[0537] In this experiment, the affinity constants of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ for C1q were measured using the Fortebio Octet molecular interaction instrument to evaluate the CDC activity of each antibody.

[0538] The affinity constants of the corresponding antibodies for C1q were determined using the Fortebio Octet molecular interaction instrument. The experimental method is briefly described as follows: Sample dilution buffer consisted of 0.02% Tween-20 and 0.1% BSA (pH 7.4) in PBS. 50 μg / mL of antibody was immobilized on a FAB2G sensor at a height of approximately 3.0 nm. The sensor was equilibrated in the buffer for 60 seconds. The immobilized antibody then bound to the antigen C1q (Sigma, Cat. No. C1740-1MG) at concentrations of 20, 10, 5, 2.5, 1.25, and 0.625 nM for the 45A5G10-HZ antigen, and 500, 250, 125, 62.5, 31.25, and 15.625 nM for the 45A5G10-HZ-B81 (DAR8) antigen. Dissociation of the antibody and antigen in the buffer was then allowed to proceed for 60 seconds. The sensor was regenerated using 10 mM glycine, pH 1.7, for 5 s, repeated three times. The data were analyzed using Data Analysis 11 to obtain affinity constants.

[0539] The results of affinity constant determination for C1q with 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ are shown in Table 19 below.

[0540] Table 19. Kinetic parameters for binding of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ to C1q

[0541] 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 to C1q was effectively eliminated after it was coupled to the toxin.

[0542] 4.5 Forte Bio's ADCC Activity Assay for Anti-c-Met Antibodies and ADCs

[0543] The ADCC effect refers to the direct killing of target cells by effector immune cells that recognize the Fc segment of antibodies bound to target cell antigens through Fc receptors (FcR) expressed on their surface.

[0544] ADCC activity of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ against MKN45 cells (from Sichuan Sibowo) expressing c-MET antigen was tested using the following method:

[0545] Weigh 0.5 g of BSA and add it to 50 mL of RPMI1640 basal medium until fully dissolved. Filter through a 0.22 μM microporous filter to obtain the sample diluent (RPMI1640 + 1% BSA), which is prepared and used immediately. The day before the experiment, target cells (MKN45) in the exponential growth phase were digested with 0.25% trypsin in a biosafety cabinet. After preparing a single-cell suspension, count the cells using a cell counter and adjust the cell density to 2 × 10 cells using RPMI1640 + 10% FBS medium. 5 Cells / mL, 100 μL / well, added to the cell culture plate, incubated in a 37°C carbon dioxide cell culture incubator overnight, and then the supernatant was discarded for later use. The effector cells (Jurkat-NFAT-CD16a, from Wuhan Taituozhong Biotechnology) in the exponential growth phase were collected and counted using a cell counter. The cell density was adjusted to 4×10 with RPMI1640 basal medium. 6 10 cells / mL, 50 μL / well, and added to the experimental 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-CD16 cell suspensions were mixed and the cell density was adjusted with RPMI1640 basal medium to a final density of 4×10 Daudi cells. 5 The final density of Jurkat-NFAT-CD16a cells was 4×10 6 50 μL / well of each 2× test substance dilution was added to the positive control wells of the assay plate. Each test substance was diluted to a 2× concentration (i.e., 40,000 ng / mL) using sample diluent, and then further diluted in a 5-fold serial series for a total of eight concentrations. Rituximab was diluted in a 7-fold serial series for a total of eight concentrations. According to the assay plate layout, 50 μL of each 2× test substance dilution was added to each well. The cell culture plate was placed on a microplate shaker at 500 rpm / min and mixed for 5 minutes. The 96-well plate was then incubated in a 37°C CO2 incubator for approximately 6 hours. After incubation, 50 μL / well of pre-thawed and room temperature Bio-Glory one-step reagent (from Adamas Life Sciences, Cat. No. RA-GL04) was added to each well of the cell culture plate. The mixture was shaken at room temperature for 10 minutes, and the bioluminescence signal was measured using a microplate reader.

[0546] The results of ADCC activity detection of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ on MKN45 cells expressing c-MET antigen are shown in FIG17 .

[0547] The results showed that in the mixed culture system of MKN45 and JurkatNFAT-CD16a, at the same dose level, the ADCC activity induced by 45A5G10-HZ-B81 (DAR8) was significantly lower than that of 45A5G10-HZ.

[0548] The results showed that 45A5G10-HZ had a weak ADCC effect, while 45A5G10-HZ-B81 (DAR8) had no obvious ADCC effect.

[0549] 4.6 Forte Bio's CDC Activity Assay for Anti-c-Met Antibodies and Their ADCs

[0550] The CDC effect is achieved by antibodies binding to corresponding antigens on the cell membrane surface and simultaneously binding to complement C1q, activating the classic pathway of complement-dependent cytotoxicity, and then forming a membrane attack complex, thereby exerting a lytic effect on target cells.

[0551] The CDC activity of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ on MKN45 cells expressing c-MET antigen was detected using the following method:

[0552] Weigh 0.5 g of BSA and add it to 50 mL of RPMI1640 basal medium to fully dissolve it. Filter it through a 0.22 μM microporous filter to obtain the sample diluent (RPMI1640 + 1% BSA), which is prepared and used immediately. Target cells (MKN45, Daudi) in the exponential growth phase are taken and prepared into a single-cell suspension. Count the cells using a cell counter and adjust the MKN45 cell count to 2 × 10 cells using RPMI1640 basal medium. 5 The Daudi cell density was adjusted to 6×10 cells / mL using RPMI1640 basal medium. 5 / mL, pipette and mix thoroughly, then add 40μL of cell suspension to each well of the cell culture plate according to the experimental plate layout. Take 1 bottle of freeze-dried guinea pig serum complement (from BERSEE item number BM361Y), add 1mL of DMEM basal medium to re-dissolve, and the complement concentration at this time is 100%. Take 1000μL of 100% complement and add it to 1000μL of DMEM basal medium and mix well. The complement concentration at this time is 50%. Add 20μL of 50% serum complement to each experimental well of the cell culture plate to make the final complement concentration in the well be 10%. Use sample diluent to dilute each sample to a 2.5× concentration (i.e. 250000ng / mL), and then further dilute each sample in a 3-fold series gradient for a total of 12 concentrations. Add 40μL of each test dilution to each well. Place the cell culture plate on a microplate shaker at 500 rpm for 5 minutes, then incubate in a 37°C CO2 incubator for approximately 6 hours. After incubation, add 50 μL / well of Cell Titer Turbo 2.0 reagent, previously melted and equilibrated to room temperature, to each well of the cell culture plate. Shake at room temperature for 10 minutes, and measure the bioluminescence signal using a microplate reader.

[0553] The results of the CDC activity assay of 45A5G10-HZ-B81(DAR8) and 45A5G10-HZ on MKN45 cells expressing c-MET antigen are shown in Figure 18. The results showed that in the presence of complement, at all dose levels, neither 45A5G10-HZ-B81(DAR8) nor 45A5G10-HZ mediated a CDC effect.

[0554] 4.7: Anti-c-MET antibodies cross-detect with human, monkey, rat, and mouse cMet

[0555] Antigen proteins human c-MET (purchased from ACRO Biosystems, product number MET-H5227), cynomolgus c-MET (purchased from ACRO Biosystems, product number MET-C52H9), rat c-MET (purchased from Sino biological, product number 80004-R08H), and mouse c-MET (purchased from Sino biological, product number 50622-M08H) were diluted in CBS at 1 μg / mL and then coated with antigen. The cells were then blocked with 2% BSA (in PBS) at 37°C for 1.5 hours. Serial dilutions of 45A5G10-HZ-B81 (DAR8) and 45A5G10-HZ were added (starting at 1 μg / mL, 4-fold dilution, 8 concentration points) and incubated at 37°C for 1 hour. HRP-labeled secondary antibodies (Goat Anti-Human IgG, Monkey ads-HRP, purchased from Southern Biotechnology) were added. Biotech, catalog number F4522-V172E), incubate at 37°C for 1 hour, add TMB substrate for color development, and read the OD value of each well at a wavelength of 450 nm (reference wavelength 630 nm) after stopping with the stop solution;

[0556] The experimental results are shown in Table 20.

[0557] Table 20. Species Cross-Reactivity of 45A5G10-HZ or 45A5G10-HZ-B81 (DAR8)

[0558] 45A5G10-HZ or 45A5G10-HZ-B81 (DAR8) specifically binds to human c-MET and monkey c-MET. The EC50 values ​​for 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 ​​for 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.

[0559] The results showed that the binding ability of 45A5G10-HZ-B81 (DAR8) to c-MET antigen of different species was similar to that of 45A5G10-HZ.

[0560] Example 5. In vivo evaluation of anti-human cMet-ADC

[0561] 5.1 Efficacy testing of anti-human cMet-ADC on non-small cell lung cancer xenografts

[0562] Test 1

[0563] To verify the in vivo efficacy of anti-human cMet ADC drugs and evaluate the anti-tumor effect of the test drugs in a subcutaneous xenograft female Balb / c Nude mouse model, 5-6 week old female Balb / c Nude mice (Vitamin B) were purchased and human non-small cell lung cancer NCI-H358 cells grown to the logarithmic growth phase were digested with EDTA and resuspended in PBS. Each mouse was subcutaneously inoculated with 5*10 6 cells, wait until the tumor grows to 100-200m 3 The drug is administered intravenously once a week at 1 mg / kg or 3 mg / kg each time.

[0564] The main observation indicators of this experiment are: 1) TGI (%), calculated as follows: 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) photos of tumor volume and tumor weight at the end of the experiment.

[0565] The experimental results, shown in Table 21 and Figure 6, show that both 45A5G10-Hz-B81(DAR8) and 55A10G6-Hz-B81(DAR8) exhibited significant tumor inhibition in mice, with even greater efficacy than ABT700-VcMMAE at a dose of 1 mg / kg. Figure 7 shows changes in mouse body weight, demonstrating that the ADC had no effect on mouse body weight throughout the dosing period.

[0566] Table 21. In vivo efficacy of anti-human cMet ADC in the NCI-H358 CDX model

[0567] Test 2

[0568] The purpose of this experiment was to evaluate the anti-tumor efficacy of the test article 45A5G10-HZ-B81 (DAR8) in the human non-small cell lung cancer cell NCI-H358 xenograft tumor model.

[0569] Female BALB / c Nu nude mice were subcutaneously inoculated with NCI-H358 cells and the tumors were grown to 180 mm. 3 About (no more than 200mm 3), 56 animals were screened and divided into 7 groups, including a Vehicle group (normal saline), a toxin (a small molecule toxin released by 45A5G10-HZ-B81 (DAR8), which can be prepared with reference to Example A1.9 of WO2022170971) 0.07 mg / kg dose group, an IgG1-B81 (isotype control antibody ADC, prepared with reference to IgG1-ADC-07 of Example 4.6.1 of WO2022170971) 3 mg / kg dose group, and 45A5G10-HZ-B81 (DAR8) 0.3, 1, and 3 mg / kg dose groups, with 8 animals in each group. Each group of animals was administered by tail vein injection once a week (QW) for 3 consecutive weeks. The day of grouping was set as the first day of the experiment (Day 1), and drug administration began on Day 1. After the last tumor measurement (Day 22, 22 days after the first dose), the animals were sacrificed to isolate the tumors and weigh them.

[0570] As shown in Table 22 and Figure 13 , the tumor volumes of animals in the 45A5G10-HZ-B81 (DAR8) 0.3, 1, and 3 mg / kg dose groups were significantly lower than those 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 dose. The tumor volume of animals in the IgG1-B81 3 mg / kg group was significantly lower than that in the Vehicle group (P < 0.01), with a tumor inhibition rate (TGI) of 49.3%. These results demonstrate that the anti-c-Met antibody-drug conjugates and their drug-linkers (toxin-linkers) provided herein achieve anti-tumor effects without requiring cellular endocytosis of the antibody or positive antigen expression on tumor cells.

[0571] The results of animal weight measurement are shown in Figure 14. At the end of the experiment (Day 22), the average body weight and body 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).

[0572] Table 22 In vivo efficacy of anti-human cMet ADC in NCI-H358 transplant tumor model

[0573] 5.2 Efficacy testing of anti-human cMet-ADC in human colon cancer CDX model

[0574] A similar approach was used to evaluate the anti-tumor effect of the anti-cMet ADC test drug in a female BALB / c nude mouse model of subcutaneous xenografts of human colon cancer SW480 cells.

[0575] The experimental results, shown in Table 23 and Figure 8, show that both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) exhibited significant tumor inhibition against tumor cells that underexpress human cMet in mice. At a dose of 5 mg / kg, the TGI was significantly higher than that of ABT700-VcMMAE (DAR4). Figure 9 shows changes in mouse body weight, demonstrating that the ADC had no effect on mouse body weight throughout the dosing process.

[0576] Table 23. In vivo efficacy of anti-human cMet ADC in the SW480 CDX model

[0577] 5.3 Efficacy testing of anti-human cMet-ADC in human colorectal adenocarcinoma CDX model

[0578] A similar approach was used to evaluate the anti-tumor effect of the cMet ADC candidate drug in a female BALB / c nude mouse model of subcutaneous xenografts of human colorectal adenocarcinoma NCI-H716 cell line.

[0579] The experimental results, shown in Table 24 and Figure 10, show that both 45A5G10-Hz-B81 (DAR8) and 55A10G6-Hz-B81 (DAR8) exhibited significant tumor inhibition in mice, with a significantly higher TGI at a 1 mg / kg dose than that of ABT700-VcMMAE (DAR4). Changes in mouse body weight are shown in Figure 11, demonstrating that the ADC had no effect on mouse body weight throughout the dosing process.

[0580] Table 24. In vivo efficacy of anti-human cMet ADC in the NCI-H716 CDX model

[0581] 5.4 Efficacy testing of anti-human cMet-ADC in human colorectal adenocarcinoma PDX model

[0582] The purpose of this experiment was to evaluate the anti-tumor effect of the test article 45A5G10-HZ-B81 (DAR8) in the human colorectal cancer CR5088 PDX model (from Sino-US Crown Biotechnology Co., Ltd.).

[0583] NOD / SCID mice were subcutaneously inoculated with CR5088 tumor masses (from Sino-US Crown Biotech Co., Ltd.) to establish a subcutaneous xenograft model of human colorectal cancer. The experiment was divided into three dose groups of the test drug 45A5G10-HZ-B81 (DAR8) (1 mg / kg, 3 mg / kg, and 10 mg / kg), and a vehicle control group, for a total of four groups, with 8 mice in each group. The drug was administered once a week for a total of 3 weeks. The tumor inhibition index (TGI) was calculated based on tumor volume for efficacy evaluation, and the safety was evaluated based on animal weight changes and mortality.

[0584] 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 ​​of less than 0.001.

[0585] The results of animal body weight changes are shown in FIG16 . All mice in each group did not experience severe weight loss (BWL<15%), and no mice died unexpectedly during the experiment, indicating that the mice tolerated the experiment well.

[0586] Table 25. Efficacy of anti-human cMet-ADC in human colorectal adenocarcinoma PDX model

[0587] 5.5 Efficacy testing of anti-human cMet-ADC on gastric cancer xenografts

[0588] The purpose of this experiment was to evaluate the anti-tumor efficacy of the test article 45A5G10-HZ-B81 (DAR8) in the human gastric cancer MKN45 xenograft tumor model.

[0589] Female NCG mice (Beijing Chuangmo) were subcutaneously inoculated with MKN45 cells (derived from JCBR JCRB0254) to establish a humanized gastric cancer model. The study was divided into a saline control group, a toxin-treated 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 8 mice in each group. Efficacy was evaluated based on relative tumor inhibition (TGI), and safety was assessed based on changes in animal body weight and mortality.

[0590] As shown in Table 26 and Figure 19, 45A5G10-HZ has the effect of inhibiting the growth of subcutaneous transplanted tumors of human gastric cancer MKN45. 45A5G10-HZ-B81 (DAR8) significantly inhibited the growth of subcutaneous transplanted tumors of human gastric cancer MKN45 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, injected into the tail vein once a week for three consecutive doses, with relative tumor inhibition rates (TGI) of 72%, 96%, and 98%, respectively, which were statistically significantly different from the vehicle control group. The tumor volume of animals 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%, indicating that the anti-c-Met antibody drug conjugates and drug linkers (toxin-linkers) provided by the present disclosure do not require cellular endocytosis of the antibody or positive expression of the antigen in tumor cells to achieve anti-tumor effects.

[0591] Table 26: NCI-H358 cell transplant model efficacy test data

[0592] Note: 1. Data are expressed as “mean ± standard error”.

[0593] 2. T / C% = TRTV / CRTV × 100%; TGI% = (1-T / C) × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the vehicle 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).

[0594] Example 6. Plasma Stability Test of C-MET Antibody-Drug Conjugate

[0595] The stability of 45A5G10-HZ-B81(DAR8) in plasma was evaluated by measuring the release of bioactive molecule toxin in human plasma after incubation of 45A5G10-HZ-B81(DAR8).

[0596] Experimental system information

[0597] 1. Experimental Procedure

[0598] Plasma preparation:

[0599] Thaw frozen plasma quickly at 37°C and place on ice before use.

[0600] Thawed plasma was filtered through a 0.22 μm filter membrane and used immediately after filtration.

[0601] Test drug dilution process:

[0602] Step 1: Prepare 1 mg / mL drug dilution: Add a certain volume of 26.6 mg / mL 45A5G10-HZ-B81 (DAR8) to the corresponding volume of 0.1 M PBS to prepare a 1 mg / mL drug dilution. Filter through a 0.22 μm filter membrane before use.

[0603] Step 2: Prepare stability samples: Take a certain volume of plasma or 0.1 M PBS and add drug diluent to a concentration of 100 μg / mL. Mix gently and dispense 200 μL into EP tubes (centrifuge tubes) (this operation should be performed on ice at all time points) and seal them tightly.

[0604] 0-min sample: immediately add 5 times the volume of methanol to the corresponding sample in step 2, vortex mix for 2 minutes, centrifuge (4°C, 17,000 × g) for 10 minutes, collect the supernatant, and freeze the supernatant below -60°C until testing.

[0605] The prepared stability samples were incubated at 37°C. After precipitation with five volumes of methanol 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 frozen at -60°C until further analysis. Free small molecules were detected by LC-MS / MS.

[0606] 2. Sample testing

[0607] The concentrations of small molecule drugs in all samples of each group were detected by LC-MS / MS.

[0608] 3. Data Processing

[0609] Calculation of the theoretical total concentration of free small molecules: Ctotal(toxin) = (Cdosed concentration (45A5G10-HZ-B81(DAR8)) / molecular weight 45A5G10-HZ-B81(DAR8)) × antibody coupling ratio (DAR) × toxin molecular weight

[0610] Percentage of free small molecule drug released (%) = C each time point (toxin) / C total (toxin) × 100%

[0611] The concentration data at each time point were retained to 3 significant figures, and the mean, standard deviation and release percentage were retained to 2 decimal places.

[0612] 4. Results

[0613] The concentrations and percentages of toxins released in plasma of different species at each time point are summarized in Table 27 below.

[0614] The results showed that after 504 hours of incubation at 37°C, the toxin release percentages of 45A5G10-HZ-B81 (DAR8) in PBS solution, cynomolgus monkey plasma, and human plasma were 0.58%, 0.44%, and 0.38%, respectively. With the increase in incubation time, the toxin release amount of all assayed matrices did not increase significantly (<0.6%), confirming that the ADC has good plasma stability and circulation stability. Other example ADCs of the present application also showed good plasma stability and circulation stability in the same test experiments, for example, with a toxin release percentage of less than 1%.

[0615] Although the specific embodiments of the present disclosure have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings published, and that such modifications are within the scope of protection of the present disclosure. The scope of protection of the present disclosure is given by the appended claims and any equivalents thereof.

Claims

1. An anti-c-MET antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the CDRs of the heavy chain variable region and / or the CDRs of the light chain variable region are identical to or have 1, 2, or 3 amino acid substitutions compared to the CDRs 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.

2. The anti-C-MET antibody or antigen-binding fragment thereof according to claim 1, comprising: HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 10; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 9; HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 12; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 11; HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO: 2; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO: 1; HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO:4; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO:3; 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; or HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO:8; and LCDR1, LCDR2, and LCDR3 contained in the light chain variable region set forth in SEQ ID NO:7; Preferably, the HCDR1-3 and the LCDR1-3 are defined according to the IMGT, Kabat or Chothia definition schemes.

3. An anti-C-MET antibody or 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 comprise the amino acid sequences 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, which comprise the amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; b. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively; c. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively; d. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which respectively comprise the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 53, and SEQ ID NO: 19; the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively; e. the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, which respectively comprise 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 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 comprising the amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 32, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively; g. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 32, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively; h. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 32, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 35, respectively; i. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which comprise the amino acid sequences shown in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which comprise the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; j. the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, which respectively comprise 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 k. the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 44, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively; 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 were determined according to the IMGT definition scheme; b. The heavy chain variable region comprises 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 comprises 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 were determined according to the Kabat definition scheme; c. the heavy chain variable region comprises 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 comprises 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 were determined according to the Kabat definition scheme; d. the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 53 and SEQ ID NO: 19, respectively; the light chain variable region comprises 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 were determined according to the Chothia definition scheme; e. The heavy chain variable region comprises 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 comprises 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 were determined according to the IMGT definition scheme; f. The heavy chain variable region comprises 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 comprises 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 were determined according to the Kabat definition scheme; g. the heavy chain variable region comprises 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 comprises 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 were determined according to the Kabat definition scheme; h. the heavy chain variable region comprises 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 comprises 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 were determined according to the Chothia definition scheme; i. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively; the light chain variable region comprises 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 were determined according to the IMGT definition scheme; j. The heavy chain variable region comprises 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 comprises 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 defined according to the Kabat definition scheme; or k. The heavy chain variable region comprises 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 comprises 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 were defined according to the Chothia definition scheme.

4. The anti-C-MET antibody or antigen-binding fragment thereof of any one of claims 1 to 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 comprised in the heavy chain variable region and the light chain variable region undergo 1, 2, or 3 amino acid substitutions; Preferably, the substitution is a conservative substitution.

5. The anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising 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 to 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 to 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 to 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 to SEQ ID NO: 11; (3) the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to 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 to 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 to 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 to 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 to SEQ ID NO: 5; or (6) the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 to SEQ ID NO:

7.

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

7.

7. The anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising an antibody heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from 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.

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

59.

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

10. A multispecific antibody comprising the anti-C-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

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

12. A host cell comprising the nucleic acid molecule of claim 11.

13. An immunoconjugate comprising: the anti-c-MET antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and an effector molecule, wherein: The effector molecule is coupled to the anti-c-MET antibody; Preferably, the effector molecule is selected from the group consisting of anti-tumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof. 14 . A method for in vivo and / or in vitro immunodetection or measurement of c-MET, comprising the step of contacting the anti-c-MET antibody of any one of claims 1 to 10 with a subject or a sample from a subject.

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

16. The antibody drug conjugate according to claim 15, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, characterized in that: The structure of the antibody-drug conjugate is shown in formula (I): in: Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antibody according to claim 10; D is the active drug 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 drug unit D respectively; The L is covalently linked to an amino residue or a sulfhydryl residue on the antibody Ab; Preferably, the L is covalently linked to a sulfhydryl residue on the antibody Ab; more preferably, the L is covalently linked to a sulfhydryl residue formed after the interchain disulfide bond on the antibody Ab is opened; Preferably, the active drug unit is selected from a cytotoxic agent; more preferably, the active drug unit is selected from a DNA topoisomerase inhibitor (e.g., a camptothecin-type biologically active molecule, such as camptothecin, DXD, a substituent-modified camptothecin or a substituent-modified DXD, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan) or a microtubule inhibitor (e.g., MMAF-type microtubule inhibitor, MMAE-type microtubule inhibitor).

17. The antibody drug conjugate of claim 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, wherein the peptide unit comprises 2-10 amino acid residues, wherein the amino acid residues are selected from natural amino acid residues, non-natural amino acid residues, or amino acid residues represented by AA1 or stereoisomers thereof; More preferably, the peptide unit is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide comprising at least one (e.g., one, two or three) amino acid residue represented by AA1 or a stereoisomer thereof; AA 1 The structures of the indicated amino acid residues are shown below, in: R a 、R b In, either one is H and the other is r 1 is 4; Or, R a With R b Together with the carbon atoms they are connected to, they form the 0 substituted 5-6 membered heterocycle; R m1 、R n1 are each independently selected from hydrogen, 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 optionally C 1-6 an alkyl-substituted 5-6 membered heterocyclic group; R m2 、R n2 are each independently selected from hydrogen and C 1-6 alkyl; 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 the group consisting of -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-Ala-(-Gly-Gly-Val-Ala-).

18. The antibody drug conjugate according to claim 16 or 17, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, wherein: The L is in, L1 is selected from: Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C 6-10 Aryl, 5-10 membered heteroaryl and amide (preferably selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond); Rx, Ry are independently selected from H and C 1-4 Alkyl; 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 connected to the antibody or antigen-binding fragment thereof through an S atom, and position 2 is connected to L2 or L3; L2 is absent or present. When L2 is present, L2 is selected from Each y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, the 1 position is connected to L1, and the 2 position is connected to L3; L3 is selected from Position 1 is connected to L1 or L2, and position 2 is connected to L4 or D; L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

19. The antibody drug conjugate of claim 18, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, wherein: L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3; Preferably, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2 or L3; and L2 is absent or present. When L2 is present, L2 is selected from Bit 1 is connected to L1, bit 2 is connected to L3; Preferably, L2 is absent.

20. The antibody drug conjugate of claim 19, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer or its pharmaceutically acceptable solvate, wherein: The The structure is as follows: Wherein, 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 antigen-binding fragment thereof through the S atom, and the 2 position is linked to D.

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

22. The antibody-drug conjugate of claim 16, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, its tautomer, or its pharmaceutically acceptable solvate, wherein the antibody-drug conjugate has the following structure: in, q as defined in claim 16; preferably 2, 4, 6 or 8; Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antibody according to claim 10; Preferably, the 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.

23. A population of antibody-drug conjugates comprising or consisting of the antibody-drug conjugate according to any one of claims 15 to 22, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, its tautomers, or its pharmaceutically acceptable solvates, wherein the antibody-drug conjugates have one, two or more q values; Preferably, the average DAR of the antibody-drug conjugate population is an integer or decimal selected from 1-16, 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 population of antibody-drug conjugates is selected from about 2.0, 4.0, 6.0, or 8.

0.

24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9, the multispecific antibody of claim 10, the antibody-drug conjugate of any one of claims 15-22, its stereoisomers, prodrugs, pharmaceutically acceptable salts, tautomers, or pharmaceutically acceptable solvates thereof, the nucleic acid molecule of claim 11, the immunoconjugate of claim 13, or the antibody-drug conjugate population of claim 23, and optionally one or more pharmaceutical excipients.

25. Use of the antibody-drug conjugate of any one of claims 15-22, its stereoisomers, prodrugs, pharmaceutically acceptable salts, tautomers, or pharmaceutically acceptable solvates thereof, the anti-C-MET antibody or antigen-binding fragment thereof of any one of claims 1-9, the multispecific antibody of claim 10, the nucleic acid molecule of claim 11, the immunoconjugate of claim 13, the antibody-drug conjugate population of claim 23, or the pharmaceutical composition of claim 24 in preparing a medicament for treating or preventing a disease associated with C-MET activity; Preferably, the disease associated with the activity of C-MET is a tumor associated with the activity of C-MET; Preferably, the tumor is selected from the group consisting of lung cancer (eg, non-small cell lung cancer, small cell lung cancer, or lung adenocarcinoma), colon cancer (eg, human colon adenocarcinoma), rectal cancer, gastric cancer, colorectal cancer (eg, colorectal adenocarcinoma).

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