Antibody-drug conjugate, and preparation method therefor and use thereof

Abstract

An antibody-drug conjugate targeting ADAM9. In the antibody-drug conjugate, a cytotoxic substance is coupled with a humanized antibody targeting ADAM9 via a new linker. The antibody-drug conjugate prepared using the linker exhibits good inhibitory activity on the growth of tumor cells.

Description

An antibody-drug conjugate, its preparation method and uses Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an antibody-drug conjugate, its preparation method, and its uses. Background Technology

[0002] Antibody-drug conjugates (ADCs) are targeted biological agents that conjugate target-specific monoclonal antibodies to highly cytotoxic small-molecule drugs via specific linkers. Using the monoclonal antibody as a carrier, these small-molecule cytotoxic drugs are efficiently delivered to target tumor cells in a targeted manner. Compared to chemotherapy drugs, ADCs can more precisely identify diseased cells, reduce damage to normal cells, and broaden the therapeutic window. Compared to traditional antibodies or antibody fragments, ADCs enhance therapeutic efficacy due to carrying highly active cytotoxic drugs.

[0003] Nearly 15 ADCs have been approved for market release; however, many ADCs have yielded disappointing clinical results, largely due to a lack of excellent efficacy and safety. From a design perspective, the antibody, linker, and payload have not yet formed a perfect combination, and there is still significant room for improvement in reducing on-target off-target toxicity, enhancing efficacy, and optimizing in vivo pharmacokinetics.

[0004] On the one hand, researchers are screening for more suitable targets and corresponding superior antibodies as carriers, and even performing necessary engineering modifications on the antibodies. On the other hand, more research is optimizing linker design to obtain a platform-type technology applicable to the delivery of different payloads and the conjugation of antibodies targeting different targets. Since most payloads are highly hydrophobic, and the effective payload is often released through the self-cleavage of PAB after enzymatic digestion, the hydrophobic nature of PAB leads to a significant tendency for aggregation in the entire ADC. Therefore, many existing technologies reduce aggregation and thus immunogenicity by increasing the hydrophilicity of the linker, such as by adding longer chains of PEG.

[0005] Meanwhile, studies have also shown that the toxicity of ADCs is multifaceted, including both on-target and off-target toxicity caused by the ADC as a whole, as well as systemic toxicity resulting from the formation of free toxins. Therefore, while improving hydrophilicity design, it is crucial to comprehensively balance the toxicity and efficacy of ADCs.

[0006] Therefore, there is an urgent need in this field to develop an antibody-drug conjugate that is highly hydrophilic, safe, and has excellent antitumor activity. Summary of the Invention

[0007] To address the above problems, this invention provides an antibody-drug conjugate, its preparation method, and its uses.

[0008] In a first aspect of the invention, a compound of formula (I) is provided, which is a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt.

[0009] Ab-(LD) f Formula (I)

[0010] Wherein, f is an integer or decimal ≥ 1, preferably 1.0 to 16.0, more preferably 3.0 to 8.0, and most preferably 4.0 to 8.0;

[0011] Ab represents the target-binding polypeptide, antibody, or antigen-binding fragment;

[0012] D is a small molecule drug, preferably a hydrophobic small molecule drug, more preferably a cytotoxic small molecule drug and / or a drug for treating autoimmune diseases and anti-inflammation;

[0013] L is a linker group that connects the antibody and D, and its structure is -L1-L2-L3-;

[0014] Wherein, L1 is the linking group used to connect Ab, and L1 is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; This indicates a double or single bond; * indicates the position where L1 connects to Ab. This is the location where L1 and L2 are connected;

[0015] L2 is a linking group used to connect L1 and L3.

[0016] Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3;

[0017] Each X 3 Independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds);

[0018] Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides;

[0019] X5 is C(O) or NH;

[0020] L4 is selected from the following group: And when the L4 mentioned above is In this case, X3 is an amino acid side chain;

[0021] Among them, L d L a and L b Select independently from the following groups:

[0022] In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5;

[0023] X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene -CO-, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH-, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5;

[0024] L c Selected from the following group:

[0025] y and z are each independently 0, 1, 2, 3 or 4;

[0026] R3, R4, R5, and R6 are hydrophilic chains, and each of them is independently selected from the following group:

[0027] Among them, n3, n4, n5 and n6 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0028] R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, cyclic glycosyl (such as cyclic glucosyl, cyclic mannosyl or cyclic galactosyl), linear glycosyl (such as linear glucosyl, linear mannosyl or linear galactosyl);

[0029] L3 is a linking group for connecting D, and the L3 is selected from the group consisting of AA, self-fractured structural fragments, or AA-self-fractured structural fragments.

[0030] AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds).

[0031] The fragments that break down are selected from the following group: PAB (p-aminobenzyloxycarbonyl), ),

[0032] In a preferred embodiment, L2 is a linking group modified by a moiety having a branched hydrophilic chain;

[0033] The module with branched hydrophilic chains includes at least one (preferably 2, 3, 4 or 5) branched frameworks and multiple (preferably 2, 3, 4 or 5) hydrophilic chains covalently connected to the branched frameworks.

[0034] Preferably, the moiety of the branched hydrophilic chain is selected from group L4:

[0035] in, Branched skeleton, * indicates the position where it connects to L1, and * indicates the position where it connects to the hydrophilic chain;

[0036] R3, R4, R5, and R6 are hydrophilic chains.

[0037] In another preferred embodiment, R3, R4, R5 and / or R6 connected to the same branch-type skeleton may be the same or different.

[0038] In another preferred embodiment, the branched backbone comprises a peptide formed by the condensation of at least two (preferably two, three, four, five or six) amino acids selected from the group consisting of lysine, aspartic acid, glutamic acid, glutamine, and arginine.

[0039] In another preferred embodiment, the branched backbone comprises a peptide segment formed by the condensation of at least two (preferably two, three, four, five, or six) trifunctional amino acids.

[0040] In another preferred embodiment, the amino acid of the trifunctional group is selected from the group consisting of lysine, aspartic acid, glutamic acid, glutamine, and arginine.

[0041] In another preferred embodiment, the module with branched hydrophilic chains is both hydrophobic and hydrophilic (i.e., an amphiphilic module that is both hydrophobic and hydrophilic).

[0042] In another preferred embodiment, the amphiphilic module has a hydrophobic proximal end and a hydrophilic distal end.

[0043] In another preferred embodiment, the hydrophobic proximal end is a branched skeleton, and the hydrophilic distal end is a plurality of hydrophilic chains (i.e., branched hydrophilic chains).

[0044] In another preferred embodiment, D is bonded to L via an amide bond.

[0045] In another preferred embodiment, X3 is an amino acid side chain; and the amino acid side chain is selected from the group consisting of:

[0046] In another preferred embodiment, X4 is selected from the group consisting of:

[0047] In a preferred embodiment, D is a cytotoxic small molecule drug selected from the group consisting of DNA damaging agents and microtubule inhibitors;

[0048] The DNA damaging agent is a topoisomerase inhibitor or a DNA binding agent.

[0049] Preferably, the topoisomerase inhibitor is from the group consisting of: Exatecan (DX8951), DXD, SN-38, 9-nitrocamptothecin, CPT-11, 10-hydroxycamptothecin, and doxorubicin metabolite PNU-159682.

[0050] The DNA damaging agent is selected from the group consisting of: pyrrolobenzodiazepines (PBD), Duocarmycin, Calicacin;

[0051] The microtubule inhibitors mentioned are selected from the following group: maytansine derivatives, eribulin, monomethyl alatatin-E (MMAE), monomethyl alatatin-F (MMAF), monomethyl dolastatin 10 (MMAD), tubulysin derivatives, cryptophycin derivatives, and taltobulin.

[0052] In another preferred embodiment, D is selected from the group consisting of:

[0053] In a preferred embodiment, the L d L a and L b Independently

[0054] The X1 mentioned is -C 1-8 Alkylene -NH- or -C 1-8Alkylene-CO-;

[0055] The L mentioned c for

[0056] In a preferred embodiment, R3, R4, R5, and R6 are each independently selected from the group consisting of:

[0057] Preferably, R3, R4, R5 and R6 are each independently selected from the following group:

[0058] In a preferred embodiment, the AA is selected from the group consisting of: Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Phe-Lys (phenylalanine-lysine), Ala-Ala-Asn (alanine-alanine-asparagine), D-Ala-Phe-Lys (D-alanine-phenylalanine-lysine), Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), Ala-Ala-Ala (alanine-alanine-alanine), and Val-Lys (valine-lysine).

[0059] In a preferred embodiment, the L4 is selected from the group consisting of:

[0060] In a preferred embodiment, the compound has the structure shown in formula (I-1):

[0061] The definitions of L2 and L3 are as described above.

[0062] In a preferred embodiment, the compound has the structure shown in formula (I-2):

[0063] The definitions of Ab, f, and L2 are as described above.

[0064] In another preferred embodiment, the coupling has the structure shown in formula (I-3):

[0065] The definitions of Ab, f, t, k, X3, and L4 are as described above.

[0066] In another preferred embodiment, the compound has the structure shown in formula (I-4):

[0067] The definitions of Ab, L3, f, m, p, n, s, t, k, X3, X4, and L4 are as described above.

[0068] In another preferred embodiment, the compound is selected from the group consisting of:

[0069] The definitions of Ab and f are as described above.

[0070] In another preferred embodiment, the antibody is the anti-ADAM9 antibody or its antigen-binding fragment from CN120058950A.

[0071] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein,

[0072] The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NO:7-10 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NO:11-12 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NO:13-16 or any variant thereof.

[0073] The heavy chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NO:17-19 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NO:20-22 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NO:23-24 or any variant thereof.

[0074] In another preferred embodiment, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment comprises:

[0075] (1) LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13, or,

[0076] (2) LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14, or,

[0077] (3) LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; or,

[0078] (4) LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:15;

[0079] The heavy chain variable region includes:

[0080] (1) HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23, or,

[0081] (2) HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22, or,

[0082] (3) HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21 and HCDR3 shown in SEQ ID NO:24;

[0083] (4) HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22 and HCDR3 shown in SEQ ID NO:23.

[0084] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment, wherein:

[0085] (1) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or,

[0086] (2) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or,

[0087] (3) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or,

[0088] (4) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or,

[0089] (5) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or,

[0090] (6) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or,

[0091] (7) The light chain variable region includes LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or,

[0092] (8) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or,

[0093] (9) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or,

[0094] (10) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or,

[0095] (11) The light chain variable region includes LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or,

[0096] (12) The light chain variable region includes LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or,

[0097] (13) The light chain variable region comprises LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or,

[0098] (14) The light chain variable region includes LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or

[0099] (16) The light chain variable region includes LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22 and HCDR3 shown in SEQ ID NO:23.

[0100] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment is selected from any one of a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a human antibody or its antigen-binding fragment, or a humanized antibody or its antigen-binding fragment.

[0101] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region or a variant thereof derived from the human κ chain, λ chain;

[0102] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region derived from the human κ chain;

[0103] More preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO:5.

[0104] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region or a variant thereof derived from human IgG1, IgG2, IgG3 or IgG4;

[0105] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4;

[0106] Further preferably, the anti-ADAM9 antibody or its antigen-binding fragment further includes a heavy chain constant region as shown in SEQ ID NO:6.

[0107] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment comprises a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the following sequences: SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34; and / or

[0108] The heavy chain variable regions are selected from the following sequences, or have at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:36.

[0109] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment comprises:

[0110] The light chain variable region shown in SEQ ID NO: 25 and the heavy chain variable region shown in SEQ ID NO: 26;

[0111] The light chain variable region shown in SEQ ID NO: 27 and the heavy chain variable region shown in SEQ ID NO: 28;

[0112] The light chain variable region shown in SEQ ID NO: 27 and the heavy chain variable region shown in SEQ ID NO: 26;

[0113] The light chain variable region shown in SEQ ID NO: 29 and the heavy chain variable region shown in SEQ ID NO: 26;

[0114] The light chain variable region shown in SEQ ID NO: 32 and the heavy chain variable region shown in SEQ ID NO: 33;

[0115] The light chain variable region shown in SEQ ID NO: 32 and the heavy chain variable region shown in SEQ ID NO: 31;

[0116] The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 35; or

[0117] The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 36.

[0118] In another preferred embodiment, the anti-ADAM9 antibody or its antigen-binding fragment contains a light chain selected from the sequences shown below, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with the following sequences: SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46; and / or

[0119] The heavy chains are selected from the following sequences, or have at least 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:48.

[0120] In another preferred embodiment, the anti-ADAM9 antibody comprises:

[0121] (1) The light chain shown in SEQ ID NO: 37 and the heavy chain shown in SEQ ID NO: 38;

[0122] (2) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 40;

[0123] (3) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 38;

[0124] (4) The light chain shown in SEQ ID NO: 41 and the heavy chain shown in SEQ ID NO: 38;

[0125] (6) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 45;

[0126] (7) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 43;

[0127] (8) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 47; or

[0128] (9) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 48.

[0129] A second aspect of the invention provides a compound of formula (II), or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof.

[0130] HLD Formula (II)

[0131] D is a small molecule drug, preferably a hydrophobic small molecule drug, more preferably a cytotoxic small molecule drug and / or a drug for treating autoimmune diseases and anti-inflammation;

[0132] L is -L1-L2-L3-;

[0133] L1 is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; This indicates a double or single bond; * indicates the position where L1 connects to H. This is the location where L1 and L2 are connected;

[0134] L2 is

[0135] Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3;

[0136] Each X3 is independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds);

[0137] Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides;

[0138] X5 is C(O) or NH;

[0139] L4 is selected from the following group: And when the L4 mentioned above is In this case, X3 is an amino acid side chain;

[0140] Among them, L d L a and L b Select independently from the following groups:

[0141] In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5;

[0142] X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene -CO-, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH-, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5;

[0143] L c Selected from the following group:

[0144] y and z are each independently 0, 1, 2, 3 or 4;

[0145] R3, R4, R5, and R6 are hydrophilic chains, and each of them is independently selected from the following group:

[0146] Wherein, n3, n4, n5 and n6 are each an integer from 0 to 20; preferably, they are any integers from 0 to 10.

[0147] R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, cyclic glycosyl (such as cyclic glucosyl, cyclic mannosyl or cyclic galactosyl), linear glycosyl (such as linear glucosyl, linear mannosyl or linear galactosyl);

[0148] L3 represents AA, AA-PAB, PAB, AA-other self-fracture structures or other self-fracture structures;

[0149] in,

[0150] L3 is a linking group for connecting D, and the L3 is selected from the group consisting of AA, self-fractured structural fragments, or AA-self-fractured structural fragments.

[0151] AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds).

[0152] The fragments that break down are selected from the following group: PAB (p-aminobenzyloxycarbonyl), ),

[0153] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, has the structure shown in formula (II-1):

[0154] The definitions of L2 and L3 are as described above.

[0155] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, has the structure shown in formula (II-2):

[0156] The definition of L2 is as described above.

[0157] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, has the structure shown in formula (II-3):

[0158] The definitions of t, k, X3, and L4 are as described above.

[0159] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, has the structure shown in formula (II-4):

[0160] The definitions of L3, m, p, n, s, t, k, X3, X4, and L4 are as described above.

[0161] In a preferred embodiment, D is a cytotoxic small molecule drug selected from the group consisting of DNA damaging agents and microtubule inhibitors;

[0162] The DNA damaging agent is a topoisomerase inhibitor or a DNA binding agent.

[0163] Preferably, the topoisomerase inhibitor is from the group consisting of: Exatecan (DX8951), DXD, SN-38, 9-nitrocamptothecin, CPT-11, 10-hydroxycamptothecin, and doxorubicin metabolite PNU-159682.

[0164] The DNA damaging agent is selected from the group consisting of: pyrrolobenzodiazepines (PBD), Duocarmycin, Calicacin;

[0165] The microtubule inhibitors mentioned are selected from the following group: maytansine derivatives, eribulin, monomethyl alistatin-E (MMAE), monomethyl alistatin-F (MMAF), monomethyl dolastatin 10 (MMAD), tubulysin derivatives, cryptophycin derivatives, and taltobulin;

[0166] More preferably, the D is selected from the group consisting of:

[0167] More preferably, the D is selected from the group consisting of:

[0168] In a preferred embodiment, the compound, or its salt, stereoisomer, solvate, or solvate of the salt, is selected from the group consisting of:

[0169] A third aspect of the present invention provides a linker compound having the structure shown in formula (III):

[0170] H-L1-L2-L3-H (Formula III)

[0171] L1 is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; This indicates a double or single bond; * indicates the position where L1 connects to H. This is the location where L1 and L2 are connected;

[0172] L2 is

[0173] Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3;

[0174] Each X3 is independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds);

[0175] Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides;

[0176] X5 is C(O) or NH;

[0177] L4 is selected from the following group: And when the L4 mentioned above is In this case, X3 is an amino acid side chain;

[0178] Among them, L d L a and L b Select independently from the following groups:

[0179] In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5;

[0180] X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene-CO, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH, - Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5;

[0181] L c Selected from the following group:

[0182] y and z are each independently 0, 1, 2, 3 or 4;

[0183] R3, R4, R5, and R6 are hydrophilic chains, and each of them is independently selected from the following group:

[0184] Wherein, n3, n4, n5 and n6 are each an integer from 0 to 20; preferably, they are any integers from 0 to 10.

[0185] R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6Hydroxyalkyl, cyclic glycosyl (such as cyclic glucosyl, cyclic mannosyl or cyclic galactosyl), linear glycosyl (such as linear glucosyl, linear mannosyl or linear galactosyl);

[0186] L3 is an AA-self-fractured structural segment, or an AA-self-fractured structural segment;

[0187] AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds).

[0188] The fragments selected from the following group: PAB (p-aminobenzyloxycarbonyl), ),

[0189] A fourth aspect of the present invention provides an intermediate for preparing a linker compound having the structure shown in formula (IV):

[0190] R1'-L2'-L3-D (IV)

[0191] R1' is selected from the following group: -NHFmoc, -NH2

[0192] L2' is

[0193] Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3;

[0194] Each X3 is independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds);

[0195] Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides;

[0196] X5 is C(O) or NH;

[0197] L4 is selected from the following group: And when the L4 mentioned above is In this case, X3 is an amino acid side chain;

[0198] Among them, L d L a and L b Select independently from the following groups:

[0199] In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5;

[0200] X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene-CO, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH-, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5;

[0201] L c Selected from the following group:

[0202] y and z are each independently 0, 1, 2, 3 or 4;

[0203] R3, R4, R5, and R6 are each independently selected from the following groups: H, Boc, Fmoc, Ac;

[0204] Wherein, n3, n4, n5 and n6 are each an integer from 0 to 20; preferably, they are any integers from 0 to 10.

[0205] R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyl groups, cyclic glycosyl groups (such as cyclic glucosyl, cyclic mannosyl, or cyclic galactosyl), linear glycosyl groups (such as linear glucosyl, linear mannosyl, or linear galactosyl), -OAc;

[0206] L3 is an AA-self-fractured structural segment, or an AA-self-fractured structural segment;

[0207] AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds).

[0208] The fragments selected from the following group: PAB (p-aminobenzyloxycarbonyl), ),

[0209] In a preferred embodiment, the intermediate compound is selected from the group consisting of:

[0210] A fifth aspect of the present invention provides an intermediate for preparing a linker compound having the structure shown in formula (V-1), (V-2), or (V-3):

[0211] Among them, R1 is selected from the following group: hydroxyl,

[0212] L1' is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; It can be a double bond or a single bond;

[0213] m, n, s, t, k, and p are each independently 0, 1, 2, 3, 4, 5, 6, or 7;

[0214] Each X 3 Independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds);

[0215] Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides;

[0216] X5 is C(O) or NH;

[0217] L3' is selected from the following group: AA, Ra, or AA-benzylbenzene compounds;

[0218] AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds).

[0219] Ra is selected from the following group:

[0220] Among them, L d '、L a 'and L b 'Select independently from the following group:'

[0221] In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5;

[0222] X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene -CO-, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene-NH(=NH)-NH, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5;

[0223] L c Selected from the following group:

[0224] y and z are each independently 0, 1, 2, 3 or 4;

[0225] R3', R4', R5', and R6' are each independently selected from the following groups: H, Boc, Fmoc, Ac,

[0226] Among them, n3, n4, n5 and n6 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0227] R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyl groups, cyclic glycosyl groups (such as cyclic glucosyl, cyclic mannose or cyclic galactosyl), and linear glycosyl groups (such as linear glucosyl, linear mannose or linear galactosyl).

[0228] In another preferred embodiment, the Selected from the following group:

[0229] In another preferred embodiment, the Selected from the following group:

[0230] In another preferred embodiment, the Selected from the following group:

[0231] In a preferred embodiment, the intermediate compound is selected from the group consisting of:

[0232] A sixth aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula I as described in the first aspect of the present invention, a compound of Formula II as described in the second aspect of the present invention, or a compound comprising a structure of Formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof; and

[0233] Pharmaceutically acceptable diluents, carriers, and / or excipients.

[0234] A seventh aspect of the present invention provides a pharmaceutical formulation comprising a compound of Formula I as described in the first aspect of the present invention, a compound of Formula II as described in the second aspect of the present invention, or a compound comprising the structure shown in Formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof; and

[0235] Pharmaceutically acceptable diluents, carriers, and / or excipients.

[0236] An eighth aspect of the present invention provides the use of a substance X in the preparation of a medicament for the prevention or treatment of cancer or inflammation;

[0237] Wherein, substance X is a compound of formula I as described in the first aspect of the present invention, a compound of formula II as described in the second aspect of the present invention, or a compound comprising the structure shown in formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the present invention, or a pharmaceutical preparation as described in the seventh aspect of the present invention.

[0238] Preferably, the cancer is a solid tumor or a non-solid tumor;

[0239] More preferably, the cancer is selected from the group consisting of: esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal carcinoma, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0240] In a ninth aspect, the present invention provides the use of substance X in the preparation of a medicament for the prevention or treatment of diseases related to abnormal cell activity;

[0241] Wherein, substance X is a compound of formula I as described in the first aspect of the present invention, a compound of formula II as described in the second aspect of the present invention, or a compound comprising the structure shown in formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the present invention, or a pharmaceutical preparation as described in the seventh aspect of the present invention.

[0242] Preferably, the disease associated with abnormal cell activity is cancer;

[0243] More preferably, the cancer is a solid tumor or a non-solid tumor;

[0244] More preferably, the cancer is selected from the group consisting of: esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal carcinoma, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0245] In a ninth aspect, the present invention provides the use of substance X in the preparation of a pharmaceutical product;

[0246] Wherein, substance X is a compound of formula I as described in the first aspect of the present invention, a compound of formula II as described in the second aspect of the present invention, or a compound comprising the structure shown in formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the present invention, or a pharmaceutical preparation as described in the seventh aspect of the present invention.

[0247] The drug is used to treat diseases related to target A, wherein target A is the target corresponding to Ab in substance X;

[0248] Preferably, the drug is used to treat cancer, a disease associated with target A;

[0249] More preferably, the cancer is selected from the group consisting of: esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal carcinoma, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0250] In a tenth aspect of the present invention, a method for in vitro non-therapeutic inhibition of tumor cells is provided, comprising the steps of: contacting the tumor cells with a compound of formula I as described in the first aspect of the present invention, a compound of formula II as described in the second aspect of the present invention, or a compound comprising a structure of formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the present invention, or a pharmaceutical formulation as described in the seventh aspect of the present invention.

[0251] In another preferred embodiment, the contact is carried out in an in vitro culture system.

[0252] In an eleventh aspect of the present invention, a method for preventing and / or treating tumors is provided, comprising the steps of: administering to a desired subject a therapeutically effective amount of a compound of formula I as described in the first aspect of the present invention, a compound of formula II as described in the second aspect of the present invention, or a compound comprising the structure shown in formula III as described in the third aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the present invention, or a pharmaceutical preparation as described in the seventh aspect of the present invention.

[0253] In another preferred embodiment, the object is a mammal, preferably a human.

[0254] In another preferred embodiment, the treatment is to inhibit the occurrence, growth, and / or metastasis of tumors.

[0255] In another preferred embodiment, the treatment further includes, simultaneously with, before, or after administering to the subject one or more treatments selected from the group consisting of radiation therapy, chemotherapy, biotherapy, or combinations thereof, a therapeutically effective amount of a compound of formula I as described in the first aspect of the invention, a compound of formula II as described in the second aspect of the invention, or a compound comprising the structure shown in formula III as described in the third aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or a solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the invention, or a pharmaceutical preparation as described in the seventh aspect of the invention.

[0256] In a twelfth aspect of the invention, a method for slowing tumor growth in a therapeutic subject is provided, comprising the steps of: combining an effective amount of a compound of formula I as described in the first aspect of the invention, a compound of formula II as described in the second aspect of the invention, or a compound comprising the structure shown in formula III as described in the third aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in the sixth aspect of the invention, or a pharmaceutical preparation as described in the seventh aspect of the invention, with one or more treatments selected from the group consisting of: radiation therapy, chemotherapy, biotherapy, or combinations thereof.

[0257] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0258] Figure 1 shows the antibody-drug conjugate inhibiting the proliferation of AGS cell lines in vitro.

[0259] Figure 2 shows the antibody-drug conjugate inhibiting the proliferation of the BXPC-3 cell line in vitro.

[0260] Figure 3 shows the antibody-drug conjugate inhibiting the proliferation of the NCI-H292 cell line in vitro.

[0261] Figure 4 shows the antibody-drug conjugate inhibiting the proliferation of the NCI-H292 cell line in vitro.

[0262] Figure 5 shows the antibody-drug conjugate inhibiting the proliferation of the HuCC-T1 cell line in vitro.

[0263] Figure 6 shows the antibody-drug conjugate inhibiting the proliferation of the NCI-H292 cell line in vitro.

[0264] Figure 7 shows how antibody-drug conjugates such as ADC-A inhibited tumor growth in mice with the NCI-H1975 tumor model in vivo.

[0265] Figure 8 shows how antibody-drug conjugates such as ADC-A inhibited tumor growth in BXPC-3 tumor model mice in vivo.

[0266] Figure 9 shows how antibody-drug conjugates such as ADC-A inhibited tumor growth in COLO-205 tumor model mice in vivo.

[0267] Figure 10 shows how antibody-drug conjugates such as ADC-A inhibited tumor growth in a mouse model of PANC 08.13 tumor in vivo.

[0268] Figure 11 shows how antibody-drug conjugates such as ADC1-A inhibited tumor growth in mice with the NCI-H292 tumor model in vivo.

[0269] Figure 12 shows how antibody-drug conjugates such as ADC1-A inhibited tumor growth in MKN45 tumor model mice in vivo.

[0270] Figure 13 shows how antibody-drug conjugates such as ADC2-A inhibited tumor growth in mice with the NCI-H292 tumor model in vivo.

[0271] Figure 14 shows how antibody-drug conjugates such as ADC-F inhibited tumor growth in mice with the NCI-H292 tumor model in vivo.

[0272] Figure 15 shows the effect of antibody-drug conjugate ADC-A on rat body weight in a rat toxicity test.

[0273] Figure 16 shows the changes in total antibody concentration in the blood of rats during the antibody-drug conjugate ADC-A toxicity test.

[0274] Figure 17 shows the release of antibody-drug conjugates such as ADC-A over time after cleavage by cathepsin B.

[0275] Figure 18 shows the release of antibody-drug conjugates ADC1-A and ADC2-A over time after cleavage by cathepsin B.

[0276] Figure 19 shows the release percentage over time of antibody-drug conjugates ADC1-A and ADC2-A after cleavage by cathepsin L;

[0277] Figure 20 shows the percentage of free eczema released in each sample (mouse, rat, monkey, and human plasma). Detailed Implementation

[0278] After long-term and in-depth research and extensive screening, the inventors discovered for the first time lysine, which has two amino groups and one carboxyl group. This technology can be used to construct a branched backbone with hydrophilic groups, and different forms and numbers of hydrophilic branches can be obtained through modular design. This modular design also helps to regulate the enzyme cleavage rate, thereby regulating the release rate of small molecule toxins and helping to optimize and improve the safety window of the ADC. Based on this, the inventors have developed an antibody-drug conjugate (ADC) with excellent hydrophilicity, safety, and antitumor activity.

[0279] the term

[0280] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0281] As used herein, the terms “antibody-drug conjugate”, “antibody-drug conjugate”, “antibody-drug conjugate”, and “antibody-drug conjugate” are used interchangeably to refer to the antibody-drug conjugate shown in formula (I).

[0282] As used in this article, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0283] As used in this article, the term "C" 1-4 "Alkyl" refers to a straight-chain or branched alkyl group having 1, 2, 3 or 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl or similar groups.

[0284] As used in this article, the term "C" 1-4"Alkoxy" refers to a straight-chain or branched alkoxy group having 1, 2, 3 or 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or similar groups.

[0285] As used herein, the term "haloalkyl" refers to a group obtained by substituting one or more hydrogen atoms in an alkyl group as described above with the same or different halogens. Specifically, "haloC" refers to a group obtained by substituting one or more hydrogen atoms in an alkyl group with the same or different halogens. 1-6 "alkyl" is preferably a halogenated C 1-4 Alkyl groups, examples of which include, but are not limited to: -CH2Cl, -CH2CF3, -CH2CCl3, perfluoroalkyl groups (e.g., -CF3-, -CF2CF3), etc.

[0286] As used herein, the term "hydroxyalkyl" refers to an alkyl group containing one or more hydroxyl groups, and the definition of an alkyl group is as described above. Examples of hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, etc.

[0287] As used herein, the term "cycloalkyl" refers to a monovalent saturated carbocyclic group consisting of carbon and hydrogen atoms, such as "C". 3-8 "Cycloalkyl" refers to a cycloalkyl group containing 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, preferably C14. 3-6 Cycloalkyl groups. Cycloalkyl groups can be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or similar groups, or bicyclic, such as fused rings, bridged rings or spirocyclic rings.

[0288] As used herein, the term "heterocyclic group" refers to a fully or partially saturated monocyclic, bicyclic, or polycyclic cyclic group with one or more heteroatoms selected from N, S, or O. For example, "4-7 membered heterocyclic group" refers to a group having 4-7 (e.g., 4, 5, 6, or 7) ring members. The nitrogen or sulfur atom may be oxidized, or the nitrogen atom may be quaternized. The heterocyclic group can be attached to any heteroatom or carbon residue in a ring or ring system molecule. Monocyclic heterocyclic groups include, but are not limited to: azacyclic butyl, pyrrolyl, oxacyclic butyl, pyrazolinyl, imidazolinyl, imidazoalkyl, oxazolinyl, isoxazolinyl, thiazoalkyl, isothiazolinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperylyl, hexahydroachenginyl, 4-piperidinoneyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxane, and tetrahydro-1,1-dioxothiophene, etc. Polycyclic heterocyclic groups include, but are not limited to, spirocyclic, fused-ring, and bridged-ring heterocyclic groups; wherein the spirocyclic, fused-ring, and bridged-ring heterocyclic groups involved are optionally connected to other groups by single bonds, or further cyclically linked to other cycloalkyl, heterocyclic, aryl, and heteroaryl groups by any two or more atoms on the ring.

[0289] As used in this article, the term "halogen" refers to F, Cl, Br, and I.

[0290] As used herein, the term "alkylene" refers to the group obtained by removing a hydrogen atom from an alkyl group as described above, such as methylene (-CH2-), ethylene (-CH2CH2-), etc.

[0291] In this article, the term "multiple" refers to 2, 3, 4, 5, or a positive integer greater than 5.

[0292] Unless otherwise specified, the term "amino acid" as used herein is intended to include any conventional amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine, tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, and arginine. It should be understood that this term includes D-type, L-type, and DL-type amino acids.

[0293] As used herein, the term "amino acid residue" refers to the group formed when the amino group in an amino acid molecule loses a hydrogen atom and the carboxyl group loses a hydroxyl group. For example, glycine residue refers to the -NH-CH2-CO- group, and valine residue refers to the -NH-CH(CH3)2-CO- group.

[0294] As used in this article, the term "n-peptide" refers to a fragment of amino acids linked together by peptide bonds. For example, "dipeptide" refers to a fragment of two amino acids linked together by peptide bonds. Similarly, "tripeptide" or "tetrapeptide" refers to fragments of three or four amino acids linked together by peptide bonds, respectively.

[0295] As used in this article, the term "amino acid side chain" refers to another group attached to the α-carbon atom in an amino acid molecule, in addition to the basic amino and carboxyl groups.

[0296] As used herein, the term "derivative" refers to a compound obtained by replacing, modifying, or deleting one or more atoms and / or groups from a compound.

[0297] As used herein, the term "cyclic glycosyl" refers to a cyclic monosaccharide unit containing a carboxyl or amino group, which can form an amide bond with a linker. Cyclic glycosyl can be cyclic glucosyl, cyclic mannose, cyclic galactosyl, etc.

[0298] As used herein, the term "linear glycosyl" refers to a linear monosaccharide unit containing a carboxyl or amino group, which can form an amide bond with a linker. Linear glycosyl can be linear glucosyl, linear mannose, linear galactosyl, etc.

[0299] As used herein, the term "self-fragmenting fragment" refers to a fragment that can spontaneously break into smaller fragments under specific conditions, including but not limited to PAB (p-aminobenzyloxycarbonyl), ),

[0300] Antibody

[0301] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0302] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0303] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0304] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0305] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies (such as antigen-binding fragments) or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0306] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0307] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0308] In this invention, the antibody of this invention also includes its conserved variant, which refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention.

[0309] The term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Journal of Immunology 170:4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system capable of recognizing and binding specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). Target antigens generally have numerous binding sites, also called epitopes, recognized by the CDRs of various antibodies. Antibodies that specifically bind to different epitopes have different structures. Therefore, an antigen can have more than one corresponding antibody. Antibodies comprise full-length immunoglobulin molecules or immunoactive portions of full-length immunoglobulin molecules, i.e., molecules containing antigens or portions thereof that specifically bind to a target of interest, including, but not limited to, cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may have any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species. However, preferably, immunoglobulins are derived from humans, mice, or rabbits.

[0310] An "antibody fragment" comprises a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; minibody (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4): 315-323); fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDR (complementarity-determining region); and any of the above epitope-binding fragments that bind to cancer cell antigens, viral antigens, or microbial antigens in an immune-specific manner; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0311] In this invention, the antibodies constituting the antibody-drug conjugate preferably retain their original wild-state antigen-binding ability. Therefore, the antibodies in this invention can, preferably specifically, bind to antigens.

[0312] In a preferred embodiment of the present invention, a preferred type of antibody or antibody fragment is an anti-ADAM9 antibody or its antigen-binding fragment, specifically, amino acids 206-297 of the anti-human ADAM9 recombinant protein (NCBI Reference Sequence: NP_003807.1) (SEQ ID NO:1), namely the ADAM9 extracellular domain (ADAM9-ECD, SEQ ID NO:2), with a C-terminus containing 6 His tags (SEQ ID NO:3), namely the human ADAM9-ECD-His protein (SEQ ID NO:4). The anti-ADAM9 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein...

[0313] The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NO:7-10 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NO:11-12 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NO:13-16 or any variant thereof.

[0314] The heavy chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NO:17-19 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NO:20-22 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NO:23-24 or any variant thereof.

[0315] Humanized antibody heavy and light chain variable region CDR sequences

[0316] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure comprises at least one LCDR selected from the sequences shown below: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0317] The antibody heavy chain variable region comprises at least one HCDR selected from the following sequences: SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

[0318] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13.

[0319] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14.

[0320] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16.

[0321] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15.

[0322] In some embodiments, the heavy chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0323] In some embodiments, the heavy chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21 and HCDR3 shown in SEQ ID NO:22.

[0324] In some embodiments, the heavy chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21 and HCDR3 shown in SEQ ID NO:24.

[0325] In some embodiments, the heavy chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23.

[0326] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0327] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22.

[0328] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0329] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23.

[0330] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0331] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22.

[0332] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0333] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23. In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; and the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0334] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0335] In some embodiments, the LCDR1 shown in SEQ ID NO:8, the LCDR2 shown in SEQ ID NO:12, and the LCDR3 shown in SEQ ID NO:14 according to this disclosure; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, the LCDR2 shown in SEQ ID NO:22, and the HCDR3 shown in SEQ ID NO:23; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, the LCDR2 shown in SEQ ID NO:22, and the HCDR3 shown in SEQ ID NO:23. In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to this disclosure includes: LCDR1 shown in SEQ ID NO:10, the LCDR2 shown in SEQ ID NO:11, and the LCDR3 shown in SEQ ID NO:15; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:17, the HCDR2 shown in SEQ ID NO:20, and the HCDR3 shown in SEQ ID NO:23.

[0336] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:87, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22.

[0337] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure includes: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; and the heavy chain variable region of the antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0338] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; and the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23.

[0339] Humanized antibody CDR region sequence

[0340] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to this disclosure is selected from: murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, human antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof.

[0341] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure further comprises a light chain constant region or a variant thereof derived from the human κ chain, λ chain;

[0342] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region derived from the human κ chain;

[0343] More preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO:5.

[0344] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure further comprises a heavy chain constant region or a variant thereof derived from human IgG1, IgG2, IgG3 or IgG4.

[0345] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4;

[0346] Further preferably, the anti-ADAM9 antibody or its antigen-binding fragment further includes a heavy chain constant region as shown in SEQ ID NO:6.

[0347] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34.

[0348] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the sequences shown below: SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:36.

[0349] In a preferred embodiment, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises:

[0350] The light chain variable region shown in SEQ ID NO: 25 and the heavy chain variable region shown in SEQ ID NO: 26;

[0351] The light chain variable region shown in SEQ ID NO: 27 and the heavy chain variable region shown in SEQ ID NO: 28;

[0352] The light chain variable region shown in SEQ ID NO: 27 and the heavy chain variable region shown in SEQ ID NO: 26;

[0353] The light chain variable region shown in SEQ ID NO: 29 and the heavy chain variable region shown in SEQ ID NO: 26;

[0354] The light chain variable region shown in SEQ ID NO: 30 and the heavy chain variable region shown in SEQ ID NO: 31;

[0355] The light chain variable region shown in SEQ ID NO: 32 and the heavy chain variable region shown in SEQ ID NO: 33;

[0356] The light chain variable region shown in SEQ ID NO: 32 and the heavy chain variable region shown in SEQ ID NO: 31;

[0357] The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 35; or

[0358] The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 36.

[0359] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure contains a light chain selected from the sequences shown below, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44 or SEQ ID NO:46.

[0360] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure contains a heavy chain selected from the sequences shown below, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:48.

[0361] In one specific embodiment, the anti-ADAM9 antibody or its antigen-binding fragment according to this disclosure comprises:

[0362] (1) The light chain shown in SEQ ID NO: 37 and the heavy chain shown in SEQ ID NO: 38;

[0363] (2) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 40;

[0364] (3) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 38;

[0365] (4) The light chain shown in SEQ ID NO: 41 and the heavy chain shown in SEQ ID NO: 38;

[0366] (5) The light chain shown in SEQ ID NO: 42 and the heavy chain shown in SEQ ID NO: 43;

[0367] (6) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 45;

[0368] (7) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 43;

[0369] (8) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 47; or (9) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 48.

[0370] Heavy and light chain variable region sequences of humanized antibodies Note: The underlined part indicates the monoclonal antibody CDR sequence.

[0371] The designed heavy and light chain variable region sequences were linked to the IgG1 heavy chain constant region and light chain constant region sequences. For example, the antibody light chain constant region was selected from the constant region of the human κ chain shown in SEQ ID NO:5, and the heavy chain constant region was selected from the native constant region of human IgG1 as shown in SEQ ID NO:6, resulting in the heavy and light chain sequences shown in Table 5.

[0372] Table 5. Heavy and light chain sequences of humanized antibodies, and constant region sequences of heavy and light chains. Note: Underlined lines indicate the monoclonal antibody CDR sequence, and double underlined lines indicate the monoclonal antibody constant region sequence.

[0373] Antibody preparation

[0374] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0375] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0376] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0377] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0378] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0379] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0380] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0381] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0382] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0383] drug

[0384] As used herein, "drug" refers to any compound having the desired biological activity and possessing reactive functional groups for the preparation of the conjugates described herein. The desired biological activity includes diagnosing, curing, alleviating, treating, and preventing diseases in humans or other animals. Therefore, the term "drug" refers to compounds identified in official national pharmacopoeias, as well as those confirmed by, for example, the United States Pharmacopeia of Allotherapy, the National Formulary, or any of its supplements, provided they possess the necessary reactive functional groups. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). It should be understood that as new drugs are discovered and developed, these drugs should also be included in the term "drug" in the conjugates described herein.

[0385] Drugs that can be used to constitute the ADC of the present invention include, but are not limited to: cytotoxic small molecule drugs, drugs for treating autoimmune diseases, and anti-inflammatory drugs.

[0386] The term "cytotoxic small molecule drugs" refers to substances that inhibit or prevent cell expression activity, cell function, and / or cause cell damage. This term includes radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic small molecule drugs include, but are not limited to: topoisomerase inhibitors (e.g., eczema, SN38, DXD, belotecone, gimetacin, and camptothecin derivatives), olstatins (e.g., olstatin E, olstatin F, MMAE, and MMAF), chlortetracycline, phenytoin, pyrethroids, pyrethroid A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, and tenoproglucoside. (oside), vincristine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, pseudomonad exotoxin (PE)A, PE40, abrin, abrin A chain, sarsaparilla root toxin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, kazimidox, Sapaonaria officinalis inhibitors, glucocorticoids, other chemotherapeutic agents, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioisotopes of Lu including Lu177. Antibodies can also be conjugated to anticancer prodrug activating enzymes capable of converting prodrugs into their active forms.

[0387] The preferred cytotoxic small molecule drugs of the present invention are compounds with high cytotoxicity, such as ethitecan, iribulin, monomethylauristatin, calcitrinin, maytansine, or combinations thereof; more preferably: monomethylauristatin-E (MMAE), monomethylauristatin-D (MMAD), monomethylauristatin-F (MMAF), or combinations thereof.

[0388] More preferred cytotoxic small molecule drugs of the present invention are ethitecan, MMAE or eribulin.

[0389] The drugs are not limited to the categories mentioned above, but also include all drugs that can be used in antibody-drug conjugates. In particular, those that can coordinate with the amide bond of the linker, such as cytotoxic drugs that coordinate with a basic amine group (primary or secondary amine). In a preferred embodiment, the drug forms a linker-drug conjugate by forming an amide bond with the linker portion.

[0390] connector

[0391] Based on the mechanism of drug release within cells, "linkers" or "linkers of antibody-drug conjugates" can be divided into two categories: unbreakable linkers and breakable linkers.

[0392] For antibody-drug conjugates containing unbreakable linkers, the drug release mechanism is as follows: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically cleaved in lysosomes, releasing an active molecule composed of a small drug molecule, the linker, and antibody amino acid residues. This alteration in drug molecular structure does not weaken its cytotoxicity; however, because the active molecule is charged (amino acid residues), it cannot penetrate neighboring cells. Therefore, such active drugs cannot kill neighboring tumor cells that do not express the target antigen (antigen-negative cells) (bystander effect).

[0393] Cleavable linkers, as the name suggests, can cleave within target cells to release the active drug (the small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically unstable linkers and enzyme-unstable linkers. Chemically unstable linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH value and glutathione concentration. pH-sensitive linkers are often called acid-cleavable linkers. These linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). First-generation antibody-drug conjugates mostly use these types of linkers, such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on these linkers typically have a short half-life (2-3 days). This short half-life limits the application of pH-sensitive linkers in next-generation antibody-drug conjugates to some extent.

[0394] For glutathione-sensitive linkers, also known as disulfide linkers, drug release is based on the difference between the high intracellular glutathione concentration (millimolar range) and the relatively low blood glutathione concentration (micromolar range). This is especially true for tumor cells, where low oxygen levels lead to increased reductase activity, resulting in even higher glutathione concentrations. Disulfide bonds are thermodynamically stable, thus exhibiting good stability in plasma.

[0395] Unstable enzyme linkers, such as peptide linkers, offer better control over drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (which is found in increased amounts in some tumor tissues). These peptide links are considered highly stable in plasma circulation because unsuitable extracellular pH and serum protease inhibitors typically render the proteases inactive. Given their high plasma stability and good intracellular cleavage selectivity and efficiency, unstable enzyme linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical unstable enzyme linkers include Val-Cit (VC) and Phe-Lys.

[0396] Self-releasing linkers are typically embedded between a breakable linker and the active drug, or are themselves part of a breakable linker. The mechanism of action of self-releasing linkers is that when the breakable linker breaks under suitable conditions, the self-releasing linker spontaneously rearranges its structure, thereby releasing the attached active drug. Common suicide linkers include para-aminobenzyl alcohols (PABs) and β-glucuronides.

[0397] The linkers of this invention utilize amino acids with trifunctional groups (e.g., lysine) to construct a branched backbone of hydrophilic groups, and obtain different forms and numbers of hydrophilic branches through modular design. The linkers designed in this invention offer diversity in combination forms, flexibility, and ease of synthesis. As the number of hydrophilic branches increases, the hydrophilicity of the linker also increases accordingly. Since lysine branches have a certain degree of hydrophobicity, one or more lysine residues forming a branched structure can create a hydrophilic / hydrophobic amphiphilic local microstructure, thereby synergistically forming a relatively ordered and stable hydrophilic structure with hydrophobic drugs. It is precisely this microstructure that helps regulate the enzyme cleavage rate, thereby regulating the release rate of small molecule toxins. Another strategy used in this invention to regulate the enzyme cleavage rate is to directly link the N-terminus of the enzyme-cleaved dipeptide to a hydrophilic group, while the side chain of the dipeptide is linked to the antibody terminus (e.g., LDC, LDD, and LDE). Simultaneously, the use of relatively short but multiple hydrophilic branches increases the hydrophilic surface area, reduces immunogenicity, and improves the overall pharmacokinetics of the ADC.

[0398] Linker-drug conjugate

[0399] The antibody-drug conjugates designed using this invention exhibit superior performance in multiple aspects. The linker-drug conjugates of this invention possess excellent anti-aggregation capabilities; the conjugated ADCs have high purity and low aggregate content, some below 1%, some with no detectable aggregates, and some even lower than the aggregation of the antibody itself. The linker-drug conjugates of this invention exhibit excellent antigen-binding activity, with binding activity essentially completely preserved compared to naked antibodies. The linker-drug conjugates of this invention demonstrate excellent biological activity in both in vivo and in vitro experiments. In vitro, they inhibited the proliferation of multiple cell lines, and in vivo, they efficiently inhibited the growth of multiple tumor models. Especially in some low-expression models, the efficacy was significantly superior to control positive ADCs using linker (deruxtecan) technology with the same target. Rat toxicity studies showed the in vivo tolerability of the linker-drug conjugates of this invention, therefore, the linker-drug conjugates of this invention are expected to have an excellent therapeutic safety window. Simultaneously, the linker-drug conjugates of this invention also exhibit excellent pharmacokinetics, with the total antibody showing a long half-life in rat blood. The linker-drug conjugate of this invention can regulate the release rate of toxins from enzymatic cleavage. As the linker is designed, different designed ADCs exhibited varying rates of toxin release under in vitro enzymatic cleavage conditions. The rate of toxin release is closely related to the efficacy and toxicity of the ADC, thus demonstrating the rationality of the linker design in this invention.

[0400] The antibody-drug conjugate provided by the present invention consists of an antibody, a linker, and a drug, wherein the linker is a combination of breakable linkers or a combination of non-breakable linkers.

[0401] Antibodies are globular proteins containing a series of amino acid sites that can be used for drug-linker conjugation. Due to their tertiary and quaternary structures, only solvent-accessible amino acids are available for conjugation. In fact, high-yield conjugation typically occurs at the ε-amino group of lysine residues or the thiol group of cysteine ​​residues.

[0402] The numerous lysine side chains on the surface of antibody proteins result in a large number of sites available for drug conjugation, leading to the formation of antibody-drug conjugates that are mixtures containing varying amounts of drug conjugates (drug / antibody ratio, DAR) and conjugation sites.

[0403] In this invention, the preferred antibodies are selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, nanobodies, or combinations thereof.

[0404] In this invention, a preferred class of antibodies targets ADAM9, HER2, EGFR, Claudin 18.2, Trop-2, ROR1, BCAM, AXL, CD19, CD20, CD22, CD276, c-Met, FOLR1, TF, Mesothelin, NECTIN4, CD37, GCP II, PD-L1, CD30, CD33, CD79B, CDKs, CEACAM5, TPBG, VTCN1, CDH3, CDH4, CDH6, CDH11, CDH17, PD-L1, HER3, B7H3, MUC1, B7H4, DLLL3, GPC3, PTK7, MSLN, cMET, EGFR / cMET, EGFR / HER3, EGFR / B7H3, Trop-2 / NECTIN4, Trop-2 / EGFR, and MUC1 / EGFR.

[0405] In this invention, a more preferred type of target is ADAM9, Trop-2, CDH17, PD-L1, CDH3, CDH6, B7H3 / PD-L1, EGFR / cMET, or EGFR / HER3.

[0406] Pharmaceutical Compositions and Administration

[0407] Because the antibody-drug conjugates provided by this invention can target specific cell populations and bind to cell surface-specific proteins (antigens), thereby releasing the drug into the cells in its active form through conjugate endocytosis or drug infiltration, the antibody-drug conjugates of this invention can be used to treat target diseases. The aforementioned antibody-drug conjugates can be administered to subjects (e.g., humans) in therapeutically effective amounts via appropriate routes. Subjects requiring treatment may be patients at risk or suspected of having a condition related to the activity or expression level of a specific antigen. Such patients can be identified through routine physical examinations.

[0408] Conventional methods, known to those skilled in the art of medicine, can be used to administer the pharmaceutical composition to a subject, depending on the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, such as oral, parenteral, inhalation spray, topical, rectal, nasal, oral, vaginal, or implantation. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Furthermore, it can be administered via injectable repositories, for example, using injectable or biodegradable materials and methods with 1, 3, or 6-month repositories.

[0409] Injectable compositions may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered via infusion, thereby delivering a pharmaceutical formulation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile formulations of a suitable soluble salt form of the antibody, may dissolve and administer pharmaceutical excipients such as water-based injections, 0.9% saline, or 5% glucose solutions.

[0410] When treated with the antibody-drug conjugate of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump. Other methods include various transport and carrier systems using conjugates and biodegradable polymers.

[0411] The pharmaceutical compositions of the present invention contain a safe and effective amount of the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated as solutions, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount.

[0412] The effective amount of the antibody-drug conjugate described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the antibody-drug conjugate, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the antibody-drug conjugate of this invention is administered daily at a dose of approximately 0.0001 mg to 50 mg / kg animal body weight (preferably 0.001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0413] Dosage forms of the antibody-drug conjugates of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0414] The antibody-drug conjugate of the present invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0415] When using the pharmaceutical composition, a safe and effective amount of the antibody-conjugate of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0416] The main advantages of this invention are:

[0417] 1. Currently, many ADCs use hydrophilic groups designed based on hydrophilic single chains. For example, a relatively long PEG chain (such as PEG 16 or PEG 24) is often used to achieve good hydrophilicity. However, functionalized high-purity PEG chains are often difficult to obtain, making the purification of linkers difficult. At the same time, single-chain PEG can only improve hydrophilicity in one way. In contrast, the hydrophilic group of this application adopts a branched chain structure, which can achieve excellent hydrophilicity with only a shorter PEG chain. Furthermore, due to the flexibility of the branched structure, it can be more flexibly optimized for toxins, and the drug release rate can be adjusted through structural differentiation.

[0418] 2. The backbone structure of the side chain in this application consists of multiple lysine residues. Two or more hydrophilic side chains formed by condensation: Lysine, which has two amino groups and one carboxyl group, is used to construct the backbone structure of the side chains. Different forms and numbers of hydrophilic side chains can be combined through modular design.

[0419] 3. The multi-hydrophilic branched design of this application can increase the surface area of ​​the hydrophilic groups, thereby improving the overall hydrophilicity and anti-aggregation ability of the antibody-drug conjugate (ADC) (i.e., low polymer content and high purity), thereby further improving the pharmacokinetics of the ADC in vivo.

[0420] 4. Currently, most linker-drug conjugates on the market use a single, purely hydrophilic side chain, while the side chain in this application possesses hydrophilic / hydrophobic amphiphilicity. This is because the lysine residues (-(CH2)4-) used to construct the side chain backbone have a certain degree of hydrophobicity, especially when using two or more lysine residues to form the side chain backbone, which helps to form a hydrophilic / hydrophobic amphiphilic structure and a locally ordered microstructure, thereby better masking the hydrophobic groups formed by hydrophobic cytotoxic drugs. Simultaneously, the high DAR value coupling method greatly improves the biocompatibility of the ADC and its pharmacokinetic properties in vivo. Other trifunctional amino acids besides lysine (such as glutamic acid and aspartic acid) can also serve as the backbone for constructing the side chain. The structural differences between glutamic acid, aspartic acid, and lysine can be further optimized for specific loading variations.

[0421] 5. The hydrophilic / hydrophobic amphiphilic structure of this application can combine with hydrophilic groups to regulate the rate at which cytotoxic drugs are cleaved by lysosomes, thereby further regulating the release of ADC efficacy in vivo and the control of toxicity.

[0422] 6. The antibody-drug conjugate obtained by conjugating the linker-drug conjugate of the present invention with the antibody has excellent hydrophilicity, safety and antitumor activity.

[0423] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, were generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0424] Example 1: Synthesis of Linker-Drug Conjugate LDA

[0425] (I) Preparation Method 1

[0426] 1.1 Synthesis of intermediate A3

[0427] Compound A1 (5.00 g, 20.30 mmol) and compound A2 (9.90 g, 22.33 mmol) were dissolved in a mixed solvent of tetrahydrofuran and water (410 mL, 10 / 1). The reaction mixture was reacted at 25 °C for 12 hours. After the reaction was complete, the reaction mixture was diluted with water (500 mL) and extracted with ethyl acetate (3 x 500 mL). The organic phases were combined, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography to give compound A3 (6.80 g, white solid). LCMS: (ESI)[M+Na] + =597.4.

[0428] 1 H NMR: (400MHz, CD3OD) δ4.08–4.00(m,1H),3.98–3.90(m,1H),3.27–3.10(m,2H),3.07–2.99(m,2H),1.87–1.57(m,4H),1.53–1.35(m,35H).

[0429] 1.2 Synthesis of intermediate A4

[0430] Compound A3 (5.80 g, 10.09 mmol) and N-hydroxysuccinimide (2.57 g, 22.33 mmol) were dissolved in tetrahydrofuran (180 mL), and DCC (2.29 g, 11.10 mmol) was added. The reaction mixture was reacted at 25 °C for 12 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated to give compound A4 (3.00 g, white solid). LCMS: (ESI)[M+Na] + =694.4.

[0431] 1.3 Synthesis of Intermediate A

[0432] Compound A4 (3.20 g, 4.76 mmol) and compound A5 (1.27 g, 7.15 mmol) were dissolved in a mixed solvent of tetrahydrofuran and water (55 mL, 10 / 1). The reaction solution was reacted at 25 °C for 12 hours. After the reaction was completed, the reaction solution was diluted with water and extracted with ethyl acetate (3 x 500 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography to give compound A (2.30 g, white solid).

[0433] LCMS:(ESI)[M+H] + =734.6.

[0434] 1 H NMR: (400MHz, DMSO-d6) δ12.16(s,1H),7.77(t,J=5.6Hz,2H),7.73(t,J=5.6Hz, 2H),6.82–6.62(m,3H),3.90–3.72(m,2H),3.59(t,J=6.4Hz,2H),3.48(s,4H),3 .38(t,J=6.4Hz,2H),3.26–3.14(m,2H),3.10–2.91(m,2H),2.90–2.82(m,2H),2 .43(t,J=6.4Hz,2H),1.54–1.42(m,4H),1.40–1.31(m,31H),1.30–1.20(m,4H).

[0435] 1.4 Synthesis of intermediate B3

[0436] Compound B1 (3.50 g, 9.22 mmol) was dissolved in DMF (18 mL), followed by the addition of compound B2 (5.74 g, 10.15 mmol) and DIEA (1.19 g, 9.22 mmol). The reaction mixture was reacted at 25 °C for 1 hour. After the reaction was complete, the mixture was concentrated to obtain a crude product, which was then slurried with dichloromethane and ethyl acetate to obtain compound B3 (6.00 g, pale yellow solid). LCMS: (ESI)[M+Na] + =852.4.

[0437] Synthesis of intermediate B5 (1.5)

[0438] Compound B3 (4.00 g, 4.82 mmol) was dissolved in DMF (60 mL), and compounds B4 (4.40 g, 14.46 mmol) and DIEA (1.59 mL, 9.64 mmol) were added separately. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated to obtain a crude product, which was then slurried successively with dichloromethane and ethyl acetate to obtain compound B5 (4.00 g, white solid). LCMS: (ESI)[M+H] + =995.5.

[0439] 1 H NMR: (400MHz, DMSO-d6) δ10.13(s,1H),8.32(d,J=8.4Hz,2H),8.17(s,1H),7.89(d,J=6.8Hz,2H) ,7.81–7.61(m,5H),7.60–7.48(m,3H),7.47–7.37(m,4H),7.36–7.26(m,2H),6.76(s,1H),6.00( s,1H),5.44(s,2H),5.25(s,2H),4.40(s,1H),4.34–4.16(m,4H),4.11–3.96(m,2H),3.13–2.76( m,5H),2.08–1.85(m,3H),1.75–1.49(m,5H),1.37(s,9H),1.22–1.14(m,2H),0.87–0.79(m,6H)._

[0440] 1.6 Synthesis of intermediate B6

[0441] Compound B5 (2.00 g, 2.01 mmol) was dissolved in DMF (20 mL), and then compound eczemabium methanesulfonate (1.07 g, 2.010 mmol) and DIEA (1.59 mL, 9.64 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was slowly added dropwise to stirred water (300 mL), filtered, and the filter cake was washed with a DMF and water mixture (30 mL, 1 / 5). The solid was dried at low temperature using an oil pump and dehydrated with toluene to obtain approximately 2.00 g of compound B6. LCMS: (ESI) [M+H]+ = 1291.6.

[0442] 1.7 Synthesis of intermediate B7

[0443] Compound B6 (2.20 g, 1.70 mmol) was dissolved in dichloromethane (25 mL), cooled to 0 °C in an ice bath, and TFA (5 mL) was added. The reaction mixture was reacted at 25 °C for 1 hour. After the reaction was complete, the reaction mixture was concentrated to give crude compound B7 (1.90 g, pale yellow solid). LCMS: (ESI)[M+H] + =1191.6.

[0444] 1.8 Synthesis of Intermediate B

[0445] Compound B7 (2.00 g, 1.68 mmol) was dissolved in DMF (30 mL), and then compound A (1.23 g, 1.68 mmol), DIEA (2.78 mL, 16.79 mmol), and HATU (0.77 g, 2.02 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was purified by reversed-phase chromatography [H2O (0.1% TFA) / ACN, 0% to 50%] to give compound B8 (1.00 g, pale yellow solid). LCMS: (ESI) [M / 2+H]+ = 954.2.

[0446] Compound B8 (1.00 g, 0.52 mmol) was dissolved in DCM (10 mL), cooled to 0 °C in an ice bath, and TFA (2 mL) was added. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated to give crude compound B (0.80 g, yellow solid). LCMS: (ESI)[M / 2+H] + =804.2.

[0447] 1.9 Synthesis of intermediate C2

[0448] Compound B (1.10 g, 0.69 mmol) was dissolved in DMF (30 mL), and then compound C1 (0.88 g, 2.12 mmol), DIEA (2.26 mL, 13.69 mmol), and HATU (0.81 g, 2.12 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was purified by reversed-phase chromatography to obtain compound C2 (800 mg, pale yellow solid). LCMS: (ESI) [M / 2+H] + =1396.0._

[0449] 1.10 Synthesis of Linker-Drug Conjugate LDA

[0450] Compound C2 (800 mg, 0.29 mmol) was dissolved in DMF (6 mL), and DEA (diethylamine) (0.45 mL, 4.30 mmol) was added. The reaction solution was reacted at 25 °C for 1 hour. After the reaction, the reaction solution was concentrated to obtain the target product, crude compound C3 (700 mg, pale yellow solid). This compound (700 mg, 0.27 mmol) was dissolved in DMF (8 mL), and compound C4 (92 mg, 0.30 mmol) and DIEA (0.09 mL, 0.55 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 1 hour. After the reaction, the reaction solution was purified by pre-HPLC (TFA) to obtain compound LDA (150.7 mg, pale yellow solid). LCMS: (ESI) [M / 2+H) + =1381.6.

[0451] 1 H NMR: (400MHz, DMSO-d6) δ10.01(s,1H),8.11(d,J=7.2Hz,1H),8.05(d,J=8.4Hz,1H ),7.98–7.86(m,5H),7.83–7.73(m,5H),7.64(d,J=8.8Hz,1H),7.60(d,J=8.8Hz,2 H),7.36(d,J=8.4Hz,2H),7.31(s,1H),6.99(s,2H),6.51(s,1H),5.97(t,J=5.7Hz ,1H),5.46–5.40(m,4H),5.28(s,3H),5.07(s,2H),4.42–4.33(m,1H),4.30–4.09( m,5H),3.60–3.54(m,10H),3.51–3.47(m,89H),3.44–3.40(m,8H),3.39–3.34(m,5 H),3.23(s,9H),3.21–3.15(m,3H),3.07–2.93(m,10H),2.41–2.34(m,8H),2.32–2 .25(m,5H),2.14–2.05(m,3H),2.00–1.94(m,1H),1.91–1.82(m,2H),1.69–1.54(m ,6H),1.51–1.41(m,9H),1.40–1.30(m,9H),1.28–1.13(m,10H),0.88–0.80(m,9H).

[0452] (II) Preparation Method 2

[0453] 2.1 Synthesis of intermediate C6

[0454] Compound B3 was dissolved in DCM and deprotected with TFA. The purified compound C5 was dissolved in DMF and reacted with compounds A, HATU, and DIEA in a condensation reaction. C6 was then purified by pre-HPLC.

[0455] 2.2 Synthesis of intermediate C8

[0456] C6 was dissolved in DCM and deprotected with TFA. The purified compound C7 was dissolved in DMF and reacted with compounds C1, HATU, and DIEA in a condensation reaction. C8 was then purified by pre-HPLC.

[0457] 2.3 Synthesis of intermediate C10

[0458] Compound C8 was dissolved in DMF and deprotected with DEA ​​to obtain compound C9. Compound C9 was dissolved in DMF and reacted with compound C3 under the condition of adding DIEA. Compound C10 was obtained by pre-HPLC purification.

[0459] 2.4 Synthesis of Linker-Drug Conjugate LDA

[0460] Compound C10 was dissolved in DMF and reacted with compound B4 with DIEA. After purification, compound C11 was obtained, dissolved in DMF and reacted with compound eczemab under the condition of DIEA addition. The reaction was then purified by pre-HPLC to obtain compound LDA. Example 2. Synthesis of the linker-drug (MMAE) conjugate (C12).

[0461] Compound C11 was prepared according to preparation method 2 in Example 1. Compound C11 was dissolved in DMF and reacted with compound MMAE under the condition of adding DIEA. Compound C12 was obtained by pre-HPLC purification.

[0462] Example 3. Synthesis of linker-drug (eribulin) conjugate (C13)

[0463] Compound C11 was prepared according to preparation method 2 in Example 1. Compound C11 was dissolved in DMF and reacted with compound eribulin under the condition of adding DIEA. Compound C13 was obtained by pre-HPLC purification.

[0464] Example 4. Linker-drug conjugate LDA similar to general synthesis

[0465] Compound C11 was prepared according to preparation method 2 in Example 1. Compound C11 was dissolved in DMF and reacted with a small molecule drug (such as other toxin compounds RH) under the condition of adding DIEA. The target compound was obtained by pre-HPLC purification.

[0466] Example 5. Synthesis of linker-drug conjugate LDB

[0467] 1.1 Synthesis of intermediate D3

[0468] Compound B (379 mg, 0.24 mmol) and compound D1 (336 mg, 0.83 mmol) were dissolved in DMF (10 mL), and HATU (314 mg, 0.83 mmol) and DIEA (610 mg, 4.72 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was directly purified by reversed-phase chromatography to obtain a white solid compound D2 (300 mg). LCMS: (ESI) [M / 2+H] + =1386.6.

[0469] Compound D2 (255 mg, 0.09 mmol) was dissolved in tetrahydrofuran (20 mL), cooled to 0 °C in an ice bath, and lithium hydroxide aqueous solution (1 M, 20 mL, 2.00 mmol) was slowly added. The reaction mixture (77 mg, 1.84 mmol) was reacted at 0 °C for 1 hour. After the reaction was complete, the pH of the reaction solution was adjusted to 6-7 with dilute hydrochloric acid, and the solution was lyophilized to obtain compound D3 (300 mg, yellow solid). LCMS: (ESI)[M / 2+H] + =1071.2.

[0470] 1.2 Synthesis of Linker-Drug Conjugate LDB

[0471] Compound D3 (300 mg, 0.14 mmol) was dissolved in DMF (5 mL), cooled to 0°C in an ice bath, and diethylamine (154 mg, 2.10 mmol) was slowly added. The reaction mixture was reacted at 25°C for 1 hour. After the reaction, the reaction mixture was purified by reversed-phase chromatography (0.1% TFA in H2O / CH3CN, 0% to 50%) to obtain a white solid compound D4 (100 mg). Compound D4 (100 mg, 0.05 mmol) was dissolved in DMF (5 mL), and compound C3 (19 mg, 0.06 mmol) and DIEA (13 mg, 0.11 mmol) were added sequentially. The reaction mixture was reacted at 25°C for 1 hour. After the reaction, the reaction mixture was purified by pre-HPLC (TFA) to obtain LDB (26.1 mg, pale yellow solid). LCMS: (ESI)[M+H) + =2112.97.

[0472] 1 H NMR: (400MHz, DMSO-d6) δ10.01(s,1H),8.12(d,J=7.2Hz,1H),8.05(d,J=8.8Hz,1H),7.99–7.91(m,2H),7.88–7.74(m,3H),7.71–7.54(m,6H),7. 36(d,J=8.4Hz,2H),7.31(s,1H),6.99(s,2H),6.51(s,1H),5.98(t,J=5 .6Hz,1H),5.60(s,2H),5.50–5.37(m,4H),5.33–5.22(m,3H),5.08(s,2H) ),4.84–4.30(m,10H),4.26–4.14(m,5H),4.07(d,J=2.8Hz,2H),3.97(d ,J=3.6Hz,1H),3.94–3.87(m,3H),3.66–3.43(m,19H),3.01(s,16H),2.3 8(s,3H),2.34–2.23(m,3H),2.22–2.04(m,4H),2.02–1.78(m,4H),1.76– 1.53(m,7H),1.51–1.31(m,13H),1.29–1.12(m,9H),0.94–0.75(m,9H)._

[0473] Example 6. Synthesis of linker-drug conjugate LDC

[0474] 1.1 Synthesis of intermediate E3

[0475] Compound E1 (20.00 g, 54.28 mmol) and compound E2 (20.48 g, 65.14 mmol) were dissolved in DMF (150 mL), and DIEA (17.94 mL, 108.57 mmol) was added. The reaction solution was reacted at 25 °C for 3 hours. After the reaction was completed, the reaction solution was concentrated. The crude product obtained was purified by column chromatography to give compound E3 (24.00 g, white solid). LCMS: (ESI)[M+H] + =567.8,t R =1.505._

[0476] 1.2 Synthesis of intermediate E5

[0477] Compound E3 (21.30 g, 37.52 mmol) was dissolved in DCM (300 mL), and then compound E4 (5.55 g, 45.03 mmol) and EEDQ (11.13 g, 45.03 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 18 hours. After the reaction was completed, the reaction solution was concentrated. The crude product was purified by slurrying with petroleum ether to give compound E5 (23.60 g, yellow solid). LCMS: (ESI)[M+H] + =672.8.

[0478] 1 H NMR: (400MHz, DMSO-d6)δ9.96(s,1H),8.06–7.86(m,3H),7.71–7.60(m,2H),7.57–7.49 (m,2H),7.41(t,J=7.2Hz,2H),7.32(t,J=7.2Hz,2H),7.28–7.18(m,3H),6.82–6.73(m,1 H),5.16–5.04(m,1H),4.46–4.36(m,3H),4.35–4.12(m,3H),3.88–3.78(m,1H),2.99–2. 93(m,1H),2.03–1.86(m,1H),1.73–1.55(m,2H),1.43–1.25(m,13H),0.87–0.81(m,6H).

[0479] 1.3 Synthesis of intermediate E7

[0480] Compounds E5 (6.00 g, 8.92 mmol) and E6 (8.14 g, 26.75 mmol) were dissolved in DMF (50 mL), and DIEA (5.76 g, 44.59 mmol) and DMAP (1.09 g, 8.918 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 4 hours. After the reaction was completed, the reaction solution was concentrated. The crude product obtained was purified by column chromatography to give compound E7 (5.20 g, yellow solid). LCMS: (ESI) [M+H-100] + =737.6.

[0481] 1.4 Synthesis of intermediate E8

[0482] Compound E7 (1.50 g, 1.79 mmol) and eczema (0.80 g, 1.84 mmol) were dissolved in DMF (50 mL), and DIEA (1.48 mL, 8.95 mmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction mixture was concentrated. The crude product was purified by rapid column chromatography to obtain compound E8 (500 mg, yellow solid). LCMS: (ESI)[M+H] + =1133.6.

[0483] Synthesis of intermediate E9 (1.5)

[0484] Compound E8 (500 mg, 0.44 mmol) was dissolved in DCM (5 mL), and TFA (1 mL) was added. The reaction solution was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction solution was concentrated. The crude product obtained was purified by reversed-phase chromatography to give compound E9 (390 mg, yellow solid). LCMS: (ESI)[M+H] + =1034.4.

[0485] 1.6 Synthesis of intermediate E10

[0486] Compound A (1.60 g, 2.18 mmol), HATU (1.10 g, 2.90 mmol), and DIEA (750 mg, 5.80 mmol) were dissolved in DMF (20 mL), and compound E9 (1.50 g, 1.45 mmol) was added. The reaction mixture was reacted at 25 °C for 18 hours. After the reaction was complete, most of the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by reversed-phase chromatography (220 g reversed-phase column, water / acetonitrile, acetonitrile gradient 0–85%) to obtain compound E10 (1.30 g, brown solid). LCMS: (ESI)[(M-100) / 2+H]+=825.6.

[0487] 1.7 Synthesis of intermediate E12

[0488] Compound E10 (1.30 g, 0.74 mmol) was dissolved in dichloromethane (15 mL), cooled to 0 °C in an ice bath, and trifluoroacetic acid (3 mL) was slowly added. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated to obtain a yellow oily compound E11 (1.00 g). Compound C1 (854 mg, 2.07 mmol), HATU (1.05 g, 2.76 mmol), and DIEA (713 mg, 5.52 mmol) were dissolved in DMF (10 mL), and the freshly prepared yellow oily compound E11 (1.00 g, 0.69 mmol) was added. The reaction solution was reacted at 25 °C for 18 hours. After the reaction was completed, most of the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by reversed-phase column chromatography to obtain compound E12 (1.40 g, brown solid). LCMS: (ESI) [M / 2+H] + =1317.3.

[0489] 1.8 Synthesis of Linker-Drug Conjugate LDC

[0490] Compound E12 (1.40 g, 0.53 mmol) was dissolved in DMF (14 mL), cooled to 0°C in an ice bath, and diethylamine (1 mL, 9.67 mmol) was added. The reaction mixture was reacted at 25°C for 1 hour. After the reaction, most of the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by reversed-phase column chromatography to obtain compound E13 (938 mg, brown solid). Compound E13 (480 mg, 0.20 mmol) and compound E14 (53 mg, 0.20 mmol) were dissolved in DMF (5 mL), cooled to 0°C in an ice bath, and DIEA (51 mg, 0.40 mmol) was added. The reaction mixture was reacted at 25°C for 2 hours. After the reaction, the pH of the reaction mixture was adjusted to 6 with formic acid, and most of the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by prep-HPLC (formic acid) to obtain LDC (26.5 mg, white solid). LCMS: (ESI) [M / 2+H] + =1281.5.

[0491] 1H NMR: (400MHz, DMSO-d6)δ9.95(s,1H),8.07–8.04(m,2H),7.94–7.86(m,5H),7.82–7.77(m,3H),7.60(d,J=8.8Hz,2H),7.36(d,J=8.8Hz,2H), 7.31(s,1H),6.98(s,2H),6.51(s,1H),5.45(s,2H),5.34–5.24(s,3H) ,5.07(s,2H),4.35–4.30(m,1H),4.24–4.13(m,3H),3.60–3.56(m,10H) ,3.50–3.47(m,92H),3.43–3.41(m,6H),3.38–3.35(m,2H),3.23(s,9H) ),3.20–3.17(m,2H),3.01–2.94(m,6H),2.40–2.35(m,6H),2.31–2.27 (m,4H),2.22–2.14(m,2H),2.01–1.82(m,4H),1.61–1.56(m,2H),1.47 –1.44(m,2H),1.35–1.31(m,4H),1.26–1.20(m,4H),0.90–0.82(m,9H).

[0492] (II) LDC Preparation Method 2

[0493] 2.1 Synthesis of intermediate E15

[0494] Compound E5 was dissolved in DCM and deprotected with TFA. The purified compound E14 was dissolved in DMF and reacted with compounds A, HATU, and DIEA in a condensation reaction. The resulting compound was purified by pre-HPLC to obtain E15.

[0495] 2.2 Synthesis of intermediate E17

[0496] E15 was dissolved in DCM and deprotected with TFA to remove the Boc protecting group. The purified compound E16 was dissolved in DMF and reacted with compounds C1, HATU, and DIEA in a condensation reaction. The resulting compound was purified by pre-HPLC to obtain E17.

[0497] 2.3 Synthesis of intermediate C19

[0498] Compound E17 was dissolved in DMF and deprotected with DEA ​​to obtain compound E18. Compound E18 was dissolved in DMF and reacted with compound E14 under the condition of adding DIEA. Compound E19 was obtained by pre-HPLC purification.

[0499] 2.4 Synthesis of Linker-Drug Conjugate LDC

[0500] Compound E19 was dissolved in DMF and reacted with compound B4 and DIEA. After purification, compound E20 was obtained, dissolved in DMF and reacted with compound eczemab under the condition of adding DIEA. The mixture was purified by pre-HPLC to obtain compound LDC.

[0501] Example 7. Synthesis of linker-drug (MMAE) conjugate (E21)

[0502] Compound E20 was prepared according to preparation method 2 in Example 6. Compound E20 was dissolved in DMF and reacted with compound MMAE under the condition of adding DIEA. Compound E21 was obtained by pre-HPLC purification.

[0503] Example 8. Synthesis of linker-drug (eribulin) conjugate (E22)

[0504] Compound E20 was prepared according to preparation method 2 in Example 6. Compound E20 was dissolved in DMF and reacted with compound eribulin under the condition of adding DIEA. Compound E22 was obtained by pre-HPLC purification.

[0505] Example 9. Linker-drug conjugate LDC similar to general synthesis

[0506] Compound E20 was prepared according to preparation method 2 in Example 6. Compound E20 was dissolved in DMF and reacted with a small molecule drug (such as other toxin compounds RH) under the condition of adding DIEA. The target compound was obtained by pre-HPLC purification.

[0507] Example 10. Synthesis of Linker-Drug Conjugate LDD

[0508] 1.1 Synthesis of intermediate F1

[0509] Compound E9 (510 mg, 0.49 mmol) was dissolved in DMF (5 mL), and then compound C1 (210 mg, 0.51 mmol), HATU (281 mg, 0.74 mmol), and DIEA (191 mg, 1.48 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 1 hour. After the reaction was completed, the reaction solution was purified by reversed-phase chromatography to obtain compound F1 (280 mg, yellow solid). LCMS: (ESI) [M / 2+H] + =715.0.

[0510] 1.2 Synthesis of intermediate F2

[0511] Compound F1 (280 mg, 0.20 mmol) was mixed with 3 mL of DMF and cooled to 0°C in an ice bath. Diethylamine (43 mg, 0.59 mmol) was then added. The reaction mixture was incubated at 25°C for 2 hours. After the reaction was complete, the solution was purified by reversed-phase chromatography to obtain compound F2 (150 mg, yellow solid). LCMS: (ESI) [M / 2+H] + =604.0.

[0512] 1.3 Synthesis of Linker-Drug Conjugates (LDDs)

[0513] Compound F2 (150 mg, 0.13 mmol) was dissolved in DMF (5 mL), followed by the addition of compounds E12 (37 mg, 0.14 mmol) and DIEA (81 mg, 0.63 mmol). The reaction mixture was incubated at 25 °C for 1 hour. After the reaction was complete, the reaction mixture was purified by pre-HPLC to obtain LDD (37.6 mg, pale yellow solid). LCMS: (ESI)[M+H) + =1358.6.

[0514] 1H NMR (400MHz, DMSO-d6) δ9.94(s,1H),8.04(d,J=7.2Hz,2H),7.92–7.85(m,2H),7.77(d,J=10.8Hz,1H),7.60(d,J=8.0Hz,2H) ,7.36(d,J=8.4Hz,2H),7.31(s,1H),6.99(s,2H),6.51(s,1H),5.44(s,2H),5.28(d,J=4.0Hz,3H),5.07(s,2H),4.37–4.28(m ,1H),4.24–4.18(m,1H),3.60–3.54(m,4H),3.52–3.46(m,28H),3.43–3.40(m,2H),3.23(s,3H),2.99–2.92(m,2H),2.39–2. 36(m,3H),2.32–2.27(m,2H),2.23–2.12(m,2H),2.02–1.84(m,3H),1.71–1.55(m,2H),1.44–1.18(s,5H),0.90–0.81(m,9H).

[0515] Example 11. Synthesis of linker-drug conjugate LDE

[0516] 1.1 Synthesis of intermediate G1

[0517] Compound D1 (779 mg, 1.92 mmol) was dissolved in DMF (10 mL), and HATU (551 mg, 1.45 mmol), DIEA (374 mg, 2.90 mmol), and E9 (1.00 g, 0.96 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction solution was purified by reversed-phase chromatography to obtain compound G1 (700 mg, yellow solid).

[0518] LCMS:(ESI)[M+H] + =1422.2.

[0519] 1.2 Synthesis of intermediate G2

[0520] Compound G1 (700 mg, 0.49 mmol) was dissolved in THF (29 mL), cooled to 0 °C in an ice bath, and lithium hydroxide aqueous solution (0.1 M, 29 mL, 2.94 mmol) was slowly added. The reaction solution was reacted at 0 °C for 2 hours. After the reaction was completed, the pH of the reaction solution was adjusted to 6 with 0.5 M hydrochloric acid, and the solution was lyophilized to give crude compound G2 (700 mg, yellow solid). LCMS: (ESI)[M+H] + =1212.6.

[0521] 1.3 Synthesis of intermediate G3

[0522] Compound G2 (700 mg, crude, 0.49 mmol) was dissolved in DMF (10 mL), cooled to 0 °C in an ice bath, and diethylamine (107 mg, 1.47 mmol) was added. The reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was purified by reversed-phase chromatography to give compound G3 (170 mg, yellow solid). LCMS: (ESI)[M+H] + =990.3.

[0523] 1.4 Synthesis of Linker-Drug Conjugate LDE

[0524] Compound G3 (170 mg, 0.17 mmol) was dissolved in DMF (10 mL), and then DIEA (65 mg, 0.51 mmol) and compound E12 (67 mg, 0.26 mmol) were added sequentially. The reaction solution was reacted at 25 °C for 2 hours. After the reaction was completed, the reaction solution was purified by Prep-HPLC (TFA) to obtain LDE (49.77 mg, white solid). LCMS: (ESI)[M+H] + =1141.6.

[0525] 1 H NMR: (400MHz, DMSO-d6) δ9.91(s,1H),8.17(d,J=7.6Hz,1H),8.05(d,J=8.8Hz,1H),7.92(t,J=5.6Hz,1H),7.77(d,J=10.8Hz,1H),7.61(d,J=8.4Hz,2 H),7.52(d,J=8.4Hz,1H),7.36(d,J=8.4Hz,2H),7.31(s,1H),6.98(s,2H), 6.52(s,1H),5.45(s,2H),5.34–5.24(m,3H),5.07(s,2H),4.35–4.29(m,1H ),4.26(dd,J=8.4,5.6Hz,1H),4.11(d,J=3.2Hz,1H),3.95–3.91(m,1H),3. 60–3.47(m,11H),3.16–3.05(m,2H),3.01–2.94(m,2H),2.37(s,3H),2.29( t,J=7.2Hz,2H),2.23–2.13(m,2H),2.09–2.02(m,1H),1.93–1.81(m,2H),1 .74–1.55(m,2H),1.41–1.30(m,3H),1.28–1.21(m,1H),0.90–0.81(m,9H).

[0526] Example 12. Synthesis of linker-drug conjugate LDF

[0527] 1.1 Synthesis of intermediate H1

[0528] TFA (50 mL) was added to a 200 mL solution of DCM containing A (10.4 g, 14.17 mmol), and the reaction was carried out at room temperature. After the reaction was complete, most of the solvent was removed by rotary evaporation, and the product was concentrated by an oil pump to obtain approximately 4.2 g of an oil. LCMS: [M+H]+ = 433.5.

[0529] 1.2 Synthesis of intermediate H3

[0530] H2 (14.8 g, 29.1 mmol) and DIEA (3.8 g, 29.1 mmol) were added to a 200 mL THF solution of H1 (4.2 g, 9.7 mmol) and reacted at room temperature. After the reaction was complete, 4.5 g of H3 was obtained by reverse column purification. LCMS: [M+H]+ = 1617.3.

[0531] 1.3 Synthesis of intermediate H4

[0532] B7 (210 mg, 0.14 mmol), 231 mg H3, and 65 mg HATU were dissolved in 4 mL of DMF. 295 μL of DIPEA was slowly added at 0°C. After the addition was complete, the reaction was allowed to proceed at room temperature. After the reaction was complete, the solution was purified to obtain 420 mg of crude H4. LCMS: [M / 2+1] + =1536.56.

[0533] 1.3 Synthesis of intermediate H5

[0534] H4 (420 mg) was dissolved in 4 mL of tetrahydrofuran, and 295 μL of DEA was slowly added at low temperature. After the addition was complete, the reaction was allowed to proceed at room temperature. After the reaction was complete, the solution was concentrated to dryness, and purified by reverse phase to obtain 330 mg of crude H5. LCMS: [M / 2+1] + =1425.86.

[0535] 1.4 Synthesis of Linker-Drug Conjugate LDF

[0536] H5 (330 mg, 0.11 mmol) and H6 (105 mg, 0.34 mmol) were dissolved in 4 mL of DMF. 50 μL of N-methylmorpholine was slowly added at 0 °C. After the addition was complete, the reaction was allowed to proceed at room temperature. After the reaction was complete, LDF was purified by prep-HPLC to obtain 49 mg, LCMS: [M / 2+1]. + =1522.05.

[0537] 1H NMR (400MHz, DMSO-d6) δ10.00 (s, 1H), 8.29 (d, J = 8.4Hz, 0.5H), 8.07 (dd, J = 32.0, 8. 6Hz,1.44H),7.97–7.84(m,4H),7.82–7.72(m,3H),7.60(dd,J=22.4,7.4Hz,3H),7.3 6–7.12(m,6.5H),6.99(s,1.5H),6.58(d,J=49.6Hz,1H),5.96(t,J=5.6Hz,1H),5.40 (s,2H),5.32(t,J=4.7Hz,1H),5.16–4.91(m,2H),4.68(d,J=31.4Hz,1H),4.46(dd,J =32.4,8.3Hz,3H),4.32–4.09(m,5H),3.99(t,J=16.2Hz,3H),3.82–3.65(m,2H),3. 62–3.54(m,9H),3.53–3.44(m,73H),3.44–3.40(m,6H),3.40–3.34(m,9H),3.26–3.2 2(m,12H),3.19(d,J=9.5Hz,5H),3.13–2.77(m,14H),2.66(s,1H),2.45–2.22(m,9H) ,2.05(d,J=36.0Hz,8H),1.85–1.12(m,40H),1.06–0.96(m,6H),0.90–0.71(m,22H).

[0538] Example 13. Preparation of Antibody

[0539] 1.1 Anti-ADAM9 antibody

[0540] The preparation method of the humanized antibody H03-2 against ADAM9 used in this application is as described in patent 202311598858.8. Its CDR combination and sequence information are shown in Table 1 below, its light and heavy chain variable region sequence information is shown in Table 2 below, and its heavy chain and light chain sequences are shown in Table 3.

[0541] Table 1. CDR sequences of heavy and light chain variable regions of humanized antibody H03-2

[0542] Table 2. Heavy and light chain variable region sequences of humanized antibody H03-2 Note: The underlined part indicates the monoclonal antibody CDR sequence.

[0543] Table 3. Heavy and light chain sequences, and constant region sequences of heavy and light chains of humanized antibodies. Note: A single underscore indicates the monoclonal antibody CDR sequence, and a double underscore indicates the monoclonal antibody constant region sequence.

[0544] 1.2 Anti-Trop-2 antibodies

[0545] The method for preparing the anti-Trop-2 antibody hRS7 used in this application is as described in patent US2004 / 0001825 A1.

[0546] 1.3 Anti-PD-L1 antibodies

[0547] The anti-PD-L1 antibody used in this application is atezolizumab, the sequences of which are shown in Table 4.

[0548] Table 4. Heavy and light chain sequences of atezolizumab

[0549] Example 14. Preparation of antibody-drug conjugate ADC-A

[0550] Antibody H03-2 was diluted with 1xPBS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 25°C water bath. 10 equivalents of DMSO solution of compound LDA (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-A. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC-A was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. The DAR value was determined to be 7.7 based on compositional analysis. The purified ADC-A showed a purity of 99.6% according to SEC.

[0551] Using the same method, ADC-A (DAR4) was prepared using TCEP equivalent to 2.1 antibody equivalents and 5 LDA equivalents. Its DAR value was determined to be 4.2 by compositional analysis. SEC analysis of the purified ADC-A showed a purity of 99.2%.

[0552] Example 15. Preparation of antibody-drug conjugate ADC-B

[0553] Antibody H03-2 was diluted with 20 mM EPPS, and 4 mM EDTA was added to adjust the pH to 6.0, resulting in an antibody concentration of 5.5 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 10 equivalents of compound LDB in DMSO solution (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-B. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC-B was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. The DAR value was determined to be 7.2 based on the compositional analysis. The purified ADC-B showed a purity of 97.7% according to SEC.

[0554] Example 16. Preparation of antibody-drug conjugate ADC-C

[0555] Antibody H03-2 was diluted with 1xPBS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 25°C water bath. 10 equivalents of DMSO solution of compound LDC (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-C. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC-C was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. The DAR value was determined to be 7.4 based on compositional analysis. The purified ADC-C showed a purity of 100% according to SEC.

[0556] Example 17. Preparation of antibody-drug conjugate ADC-D

[0557] Antibody H03-2 was diluted with 50 mM EPPS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 5 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of DMSO solution of compound LDD (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-D. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC-D was obtained and stored in a 20 mM histidine solution containing 5% sucrose, 0.02% Tween 80, and pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. The DAR value was determined to be 6.0 based on compositional analysis. The purified ADC-D showed a purity of 100% according to SEC.

[0558] Example 18. Preparation of antibody-drug conjugate ADC-E

[0559] Antibody H03-2 was diluted with 20 mM EPPS, and 4 mM EDTA was added to adjust the pH to 6.0, resulting in an antibody concentration of 5 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 10 equivalents of DMSO solution of compound LDE (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-E. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC-E was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. The DAR value was determined to be 7.5 based on compositional analysis. The purified ADC-E showed a purity of 95.9% according to SEC.

[0560] Example 19. Preparation of positive control antibody-drug conjugate ADC-deruxtecan

[0561] Antibody H03-2 was diluted with 50 mM EPPS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 5 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 12 equivalents of DMSO solution (final DMSO concentration 12%) of the compound deruxtecan (deruxtecan is the linker-drug conjugate of Daiichi Sankyo Enhertu ADC) were added to the antibody mixture. After 60 minutes, 12 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-deruxtecan. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugate ADC-deruxtecan was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 7.5 based on compositional analysis. The purified ADC-dereuxtecan showed a purity of 98.7% according to SEC.

[0562] Example 20. Preparation of antibody-drug conjugate ADC1-A

[0563] Anti-Trop-2 antibody hRS7 (prepared as described in patent US2004 / 0001825 A1) was diluted with 50 mM EPPS buffer, 10 mM EDTA was added, and the pH was adjusted to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution, and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of DMSO solution of LDA (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC1-A. After desalting using a desalting column (packing material: Sephadex G 25) and ultrafiltration purification, the conjugated product ADC1-A was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 7.8 based on compositional analysis. The purified ADC1-A showed a purity of 99.7% according to SEC.

[0564] Example 21. Preparation of antibody-drug conjugate ADC1-deruxtecan

[0565] Anti-Trop-2 antibody hRS7 was diluted with 50 mM EPPS buffer, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of DMSO solution of the compound deruxtecan (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC1-deruxtecan. After desalting and ultrafiltration using a desalting column (packing material: Sephadex G 25), the conjugated product ADC1-deruxtecan was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 7.9 based on compositional analysis. The purified ADC1-deruxtecan showed a purity of 99.6% according to SEC.

[0566] Example 22. Preparation of antibody-drug conjugate ADC1-PB038

[0567] The anti-Trop-2 antibody hRS7 was diluted with 50 mM EPPS buffer, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of compound PB038 (PB038 is a linker-drug conjugate of PropFang Biotech's PRO1184 ADC, commercially available from MedChemExpress) in DMSO solution (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was incubated at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC1-PB038. After desalting and ultrafiltration using a desalting column (Sephadex G 25 packing material), the conjugate ADC1-PB038 was obtained and stored in a 20 mM histidine solution at pH 5.5 containing 5% sucrose and 0.02% Tween 80. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. The DAR value was determined to be 7.9 based on compositional analysis. SEC analysis of the purified ADC1-PB038 showed a purity of 98.5%.

[0568] Example 23. Preparation of antibody-drug conjugate ADC2-A

[0569] Anti-PD-L1 antibody K042 was diluted with 50 mM EPPS buffer, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / mL. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of DMSO solution of LDA (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC2-A. After desalting and ultrafiltration using a desalting column (Sephadex G 25 packing material), the conjugated product ADC2-A was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. Compositional analysis determined the DAR value to be 7.9. The purified ADC2-A showed a purity of 99.8% according to SEC.

[0570] Example 24. Preparation of antibody-drug conjugate ADC2-deruxtecan

[0571] Anti-PD-L1 antibody K042 was diluted with 50 mM EPPS buffer, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of DMSO solution of the compound deruxtecan (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC2-deruxtecan. After desalting using a desalting column (packing material: Sephadex G 25) and ultrafiltration purification, the conjugated product ADC2-deruxtecan was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 7.8 based on compositional analysis. The purified ADC2-deruxtecan showed a purity of 99.6% according to SEC.

[0572] Example 25. Preparation of antibody-drug conjugate ADC2-PB038

[0573] Anti-PD-L1 antibody K042 was diluted with 50 mM EPPS buffer, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 22°C water bath. 13 equivalents of DMSO solution of compound PB038 (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 13 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 22°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC2-PB038. After desalting and ultrafiltration using a desalting column (Sephadex G 25 packing material), the conjugated product ADC2-PB038 was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 8.0 based on compositional analysis. The purified ADC2-PB038 showed a purity of 99.3% according to SEC.

[0574] Example 26. Preparation of antibody-drug conjugate ADC-F

[0575] Anti-ADAM9 antibody H03-2 was diluted with 50 mM EPPS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 25°C water bath. 10 equivalents of DMSO solution (final DMSO concentration 12%) of the compound LDF obtained in Example 12 were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-F. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC-F was obtained and stored in a 20 mM histidine solution containing 5% sucrose, 0.02% Tween 80, and pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. Compositional analysis determined the DAR value to be 4.4. The purified ADC-F showed a purity of 99.2% according to SEC.

[0576] Example 27. Preparation of antibody-drug conjugate ADC-vcMMAE

[0577] Anti-ADAM9 antibody H03-2 was diluted with 50 mM EPPS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 25°C water bath. 10 equivalents of DMSO solution (commercially available) containing vcMMAE (DMSO final concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C on a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC-vcMMAE. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugate ADC-vcMMAE was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 4.2 based on compositional analysis. The purified ADC-vcMMAE showed a purity of 99.4% according to SEC.

[0578] Example 28. Preparation of antibody-drug conjugate ADC2-F

[0579] Anti-PD-L1 antibody K042 was diluted with 50 mM EPPS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 25°C water bath. 10 equivalents of LDF in DMSO solution (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC2-F. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugated product ADC2-F was obtained and stored in a 20 mM histidine solution containing 5% sucrose, 0.02% Tween 80, and pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of the conjugated and unconjugated antibodies. Compositional analysis determined the DAR value to be 5.5. The purified ADC2-F showed a purity of 98.8% according to SEC.

[0580] Example 29. Preparation of antibody-drug conjugate ADC2-vcMMAE

[0581] Anti-PD-L1 antibody K042 was diluted with 50 mM EPPS, and 10 mM EDTA was added to adjust the pH to 7.0, resulting in an antibody concentration of 10 mg / ml. 6.5 equivalents of TCEP aqueous solution were added to the antibody solution and the mixture was shaken at 37°C for 2 hours. The sample was then placed in a 25°C water bath. 10 equivalents of DMSO solution of compound vcMMAE (final DMSO concentration 12%) were added to the antibody mixture. After 60 minutes, 10 equivalents of acetylcysteine ​​(NAC) were added, and the mixture was reacted at 25°C with a shaker at 30 rpm for 10 minutes to obtain the crude conjugated product ADC2-vcMMAE. After desalting and purification using a desalting column (packing material: Sephadex G 25), the conjugate ADC2-vcMMAE was obtained and stored in a 20 mM histidine solution containing 5% sucrose and 0.02% Tween 80 at pH 5.5. The reduced ADC and antibody were analyzed by RP-HPLC to determine the light and heavy chains of both conjugated and unconjugated antibodies. The DAR value was determined to be 4.8 based on compositional analysis. The purified ADC2-vcMMAE showed a purity of 98.8% according to SEC.

[0582] The parameters of the antibody-drug conjugates prepared in the above embodiments are summarized as follows:

[0583] Example 30. In vitro antitumor activity of anti-ADAM9 antibody-drug conjugate

[0584] This embodiment tested the in vitro antitumor activity of the antibody-drug conjugate against ADAM9-expressing gastric cancer cells AGS and ADAM9-expressing pancreatic cancer cells BXPC-3.

[0585] The cells used in this example were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. or Nanjing Kebai Biotechnology Co., Ltd. (both have STR identification reports) and cultured according to the corresponding instructions, including: AGS, BxPC-3. Cells in the logarithmic growth phase were seeded at a density of 2000 cells per well into 96-well cell culture plates (100 μL / well). After incubation at 37°C and 5% CO2 for approximately 24 hours, different concentrations of antibody-drug conjugates were added, with three replicates for each drug concentration, along with corresponding solvent control and blank control wells. After 5 days (120 hours), the culture medium was discarded, and 100 μL / well of complete culture medium containing 10% CCK-8 (purchased from Beyotime, CAT#C0040) was added. The cells were incubated at 37°C for 1-2.5 hours (depending on cell reactivity, until the desired color depth was reached). Cell viability (OD 450 nM) was measured for each group, and cell survival rate was calculated using the following formula: Survival rate = (OD of drug-treated cells - OD of blank cells) / (OD of control cells - OD of blank cells) × 100%. The data were analyzed using software to calculate the IC50 of each antibody-drug conjugate on different cell lines.50 value.

[0586] Experimental results showed that, in vitro, ADC-A, ADC-B, ADC-D, and ADC-E targeting ADAM9 effectively inhibited the growth of AGS and BXPC-3 tumor cells with high ADAM9 expression (Figures 1 and 2). The concentrations in the figures represent the molar concentrations of eczema. Example 31. In vitro antitumor activity of anti-TROP-2 antibody-drug conjugates.

[0587] This example tested the in vitro antitumor activity of the antibody-drug conjugate against TROP-2-expressing lung cancer cells NCI-H292. The cell culture and experimental methods were the same as in Example 30.

[0588] Experimental results showed that ADC1-A, ADC1-derutecan, and ADC1-PB038, which target TROP-2 in vitro, could effectively inhibit the growth of NCI-H292 tumor cells with high TROP-2 expression (Figure 3). The concentrations in the figure represent the concentrations of ADCs.

[0589] Example 32. In vitro antitumor activity of anti-PD-L1 antibody-drug conjugate

[0590] This example tested the in vitro antitumor activity of the antibody-drug conjugate against NCI-H292 lung cancer cells expressing PD-L1. The cell culture and experimental methods were the same as in Example 30.

[0591] Experimental results showed that ADC2-A, ADC2-derutecan, and ADC2-PB038, which target PD-L1 in vitro, could effectively inhibit the growth of NCI-H292 tumor cells with high PD-L1 expression (Figure 4). The concentrations in the figure represent the concentrations of ADCs.

[0592] Example 33. In vitro antitumor activity of antibody-drug conjugates containing MMAE as a toxin.

[0593] This example tested the in vitro antitumor activity of the antibody-drug conjugate with MMAE as the toxin against HuCC-T1 (human hepatobiliary carcinoma cells) expressing ADAM9 and against NCI-H292 (lung cancer cells) expressing PD-L1. The cell culture and experimental methods were the same as in Example 30.

[0594] Experimental results showed that both ADC-F and ADC-vcMMAE targeting ADAM9 effectively inhibited the growth of HuCC-T1 tumor cells with high ADAM9 expression in vitro (Figure 5), where the concentration in the figure represents the concentration of ADC. Additionally, both ADC2-F and ADC2-vcMMAE targeting PD-L1 effectively inhibited the growth of NCI-H292 tumor cells with high PD-L1 expression in vitro (Figure 6), where the concentration in the figure represents the concentration of ADC.

[0595] Example 34. Detection of relative binding activity of antibody-drug conjugates

[0596] The binding activity of the antibody-conjugate to human ADAM9 protein was detected by enzyme-linked immunosorbent assay (ELISA). The His Tag-labeled Human ADAM9 antigen was bound to the ELISA plate and incubated at 2–8°C for 12–18 hours, followed by BSA blocking (incubation at 20–28°C for 60–90 minutes).

[0597] Add 100 μL of serially diluted sample to each well of the ELISA plate and incubate at 20–28°C for 60–90 minutes. Wash away the free sample. Add 100 μL of detection working solution (AffiniPure F(ab')2Fragment Donkey Anti-Human IgG(H+L)) and incubate at 20–28°C for 60–90 minutes. After incubation, wash away the free detection working solution, add TMB for color development, and terminate the reaction with acid. Measure the absorbance at 450 / 650 nm using an ELISA reader as the detection / reference value.

[0598] Four-parameter fitting was performed using SoftMax software to plot the dose-response curve between absorbance and sample concentration. EC was used for further analysis. 50 (Half-maximal effective concentration) The relative binding activity is calculated using the following formula: Relative binding activity of sample = EC of standard. 50 ÷Sample EC 50 ×100%, the results are shown in the table below.

[0599] The experimental results showed that, compared with the control ADC-deruxtecan, ADC-A, ADC-B, ADC-C, ADC-D, and ADC-E all exhibited unexpectedly superior relative binding activity. For example, the relative binding activity of ADC-A was 50% higher than that of ADC-deruxtecan.

[0600] Example 35. In vivo antitumor activity of anti-ADAM9 antibody-drug conjugate

[0601] NCI-H1975, BXPC-3, and Colo-205 cells were cultured in vitro in monolayers and passaged. Cells were harvested when they reached the exponential growth phase. 5.0 × 10⁶ cells were then cultured. 6 One tumor cell was suspended in 0.1 ml of a 1:1 mixture of PBS and Matrigel and inoculated into the right scapula of five nude mice (P1 generation). Tumors were cultured until they reached 500-800 mm in size. 3 At that time, the tumor-bearing mice were euthanized by CO2 anesthesia, and the tumor was cut into 20-30mm pieces. 3 Small tumor fragments were inoculated into a new batch of nude mice (P2 generation). The antitumor activity of the test product was evaluated using stable passaged tumor tissue. Seven days after inoculation, the average tumor volume reached approximately 150 mm². 3 Mice (n=5) were randomly grouped according to tumor volume and drug administration was initiated. In the NCI-H1975 and BXPC-3 models, ADC-A, ADC-B, and ADC-E were administered twice weekly at a dose of 0.1 mg / kg of toxin (ezeticon). In the Colo-205 model, ADC-A and ADC-E were administered once weekly at a dose of 0.2 mg / kg of toxin (ezeticon).

[0602] After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were monitored: animal activity, food and water intake, weight changes (measured twice weekly), and any abnormalities in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. Tumor volume was calculated as follows: tumor volume (mm²) 3 )=1 / 2×(a×b 2 (Where a represents the major axis and b represents the minor axis). Around day 35 after tumor inoculation, tumors were harvested from all mice, weighed, and photographed. The tumor inhibition rate (TGI) at the end of the experiment was calculated as (1 - average tumor weight in the treatment group / average tumor weight in the control group) × 100%.

[0603] The antibody-drug conjugates ADC-A, ADC-B and ADC-E targeting ADAM-9 of the present invention all showed significant antitumor effects on ADAM9-expressed BXPC-3 (Figure 8).

[0604] In particular, when using the same antibody, each linker-drug conjugate of the present invention achieved better tumor-suppressing effects than the linker-drug conjugate of the Daiichi Sankyo Enhertu ADC, indicating that the linker-drug conjugate of the present invention contributes to the improvement of this inhibitory effect.

[0605] The antibody-drug conjugates ADC-A and ADC-E targeting ADAM-9 of the present invention both showed significant antitumor effects on Colo-205 (Figure 9).

[0606] In particular, when using the same antibody, each linker-drug conjugate of the present invention achieved better tumor-suppressing effects than the linker-drug conjugate of the Daiichi Sankyo Enhertu ADC, indicating that the linker-drug conjugate of the present invention contributes to the improvement of this inhibitory effect.

[0607] In another in vivo efficacy study, the antibody-drug conjugate ADC-A (DAR4), targeting ADAM-9 with a DAR value of 4, demonstrated excellent tumor inhibition in PANC 08.13 (human pancreatic adenocarcinoma cells). A single dose of 3 mg / kg (ADC dose) achieved 100% tumor inhibition (Figure 10).

[0608] Example 36. In vivo antitumor activity of anti-TROP-2 antibody-drug conjugate

[0609] This example tested the in vivo antitumor activity of the antibody-drug conjugate against TROP-2-expressing lung cancer cells NCI-H292 and gastric cancer cells MKN45. The mouse tumor model was established using tumor fragment inoculation, and the inoculation and experimental methods were the same as in Example 35. The NCI-H292 model was administered a single dose of 3 mg / kg. The MKN45 model was administered a three-weekly dose of 5 mg / kg of ADC.

[0610] The statistical curves of the tumor-suppressing activity of the NCI-H292 model are shown in Figure 11. Among them, NCI-H292 is a TROP-2 high-expression model, and 100% tumor inhibition rate was achieved with a single dose of ADC1-A, ADC1-deruxtecan and ADC1-PB038.

[0611] The statistical curve of the tumor-suppressive activity of the MKN45 model is shown in Figure 12. MKN45 is a TROP-2 low-expression model, and ADC1-A showed superior tumor-suppressive effect compared with the positive control ADC1-deruxtecan and ADC1-PB038 after three doses.

[0612] The results showed that in the high-expression model, when using the same antibody, the linker-drug conjugate of the present invention achieved a similar tumor-suppressing effect to the linker-drug conjugate of Daiichi Sankyo Enhertu ADC and the linker-drug conjugate of Pufang Bio PRO1184 ADC. Moreover, in the Trop-2 low-expression model, the linker-drug conjugate of the present invention achieved a better tumor-suppressing effect. This result indicates that, compared with the control, ADC1-A has a wider adaptability to the Trop-2 target.

[0613] Example 37. In vivo antitumor activity of anti-PD-L1 antibody-drug conjugate

[0614] This example tested the in vivo antitumor activity of the antibody-drug conjugate against NCI-H292 lung cancer cells expressing PD-L1. Cell culture and experimental methods were the same as in Example 35. The NCI-H292 model was administered the ADC at a single dose of 10 mg / kg.

[0615] The statistical curve of the tumor-suppressive activity of the NCI-H292 model is shown in Figure 13. NCI-H292 is a PD-L1 low-expression model, and ADC2-A showed superior tumor-suppressive effects compared with the positive controls ADC2-deruxtecan and ADC2-PB038 after a single dose.

[0616] Example 38. In vivo antitumor activity of MMAE-based antibody-drug conjugates.

[0617] This example tested the in vivo antitumor activity of an antibody-drug conjugate containing MMAE as a toxin against NCI-H292 lung cancer cells expressing ADAM9. Cell culture and experimental methods were the same as in Example 35. The NCI-H292 model was administered 0.1 mg / kg twice weekly.

[0618] The statistical curve of the tumor-suppressive activity of the NCI-H292 model is shown in Figure 14. NCI-H292 is a model with low expression of ADAM9. ADC-F showed superior tumor-suppressive effect compared to ADC-vcMMAE after two doses.

[0619] The above results demonstrate that, in the ADAM9 low expression model, the linker-drug conjugate of the present invention can achieve better tumor suppression effect.

[0620] Example 39. Rat toxicity test and toxicokinetics of antibody-drug conjugates

[0621] SD rats (one male and one female, 6-9 weeks old, weighing 180-240g) were administered a blank solvent or antibody-drug conjugate via slow tail vein injection, once a week for three weeks, at a dose of 160mg / kg. The animals were weighed before administration and twice a week after administration, and their condition was observed. Simultaneously, 0.3mL of blood was collected from the jugular vein at different time points (5min, 4h, 24h, 48h, 72h, 120h, and 168h) after the first and third administrations for the detection of toxicogenized total antibodies. The total antibody content in rat serum samples was detected using an enzyme-linked immunosorbent assay (ELISA). The antigen was bound to a solid-phase support. During detection, the antibody-drug conjugate in the test serum was incubated with the antigen bound to the solid-phase support. After washing, the working solution was added, followed by TMB colorimetric development. The total antibody content in the sample was determined by quantitatively detecting the amount of the colorimetric product.

[0622] At a dose of 160 mg / kg, ADC-A showed good tolerability, with weight loss following each administration. With three consecutive weekly administrations, no animal deaths occurred, and weight slowly recovered after the end of the administration period, as shown in Figure 15.

[0623] Simultaneously, toxicokinetic analysis of the total anti-antibody concentration in the blood showed the excellent pharmacokinetics of ADC-A. The high dosing frequency resulted in a significantly higher total anti-antibody concentration after the third dosing compared to the first dosing, as shown in Figure 16.

[0624] Example 40. Enzyme cleavage assay of ADC

[0625] 75 μL of 80 mM cysteine, 35 μL of 0.1% Brij, and 10 μL of cathepsin B (50 μg / mL) (Yiqiao Shenzhou, 10480-H08H) were added sequentially to EP tubes. The tubes were incubated at 37°C for 15 minutes. Then, 85 μL of substrate (pH 5.0) of each ADC (ADC-A, ADC-B, ADC-C, ADC-D, ADC-E, and ADC-deruxtecan) (10 mg / mL) were added, mixed well, and placed in a sample pan preheated to 37°C for enzymatic digestion. At different time points, the release of free eczema or DXD was detected by RP-HPLC, and the percentage of toxin released was calculated. The results are shown in Figure 17. Here, ADC-B showed the fastest enzymatic cleavage rate, releasing 3.2 times (24 hours) and 2.7 times (42 hours) more toxin than ADC-A.

[0626] In another experiment, ADC1-A, ADC1-deruxtecan, ADC1-PB038, ADC2-A, ADC2-deruxtecan, and ADC2-PB038 were added under the same experimental conditions. The percentage of free eczema or DXD released after enzyme lysis at different time points is shown in Figure 18. The enzyme lysis rate of ADC1-PB038 was significantly faster than that of ADC1-A, with the amount of toxin released being 2.5 times (24 hours) and 2.0 times (48 hours) that of ADC1-A. Similarly, the enzyme lysis rate of ADC2-PB038 was significantly faster than that of ADC2-A, with the amount of toxin released being 1.7 times (24 hours) that of ADC2-A.

[0627] In another experiment, under the same conditions, cathepsin L was used to cleave ADC2-A, ADC2-deruxtecan, and ADC2-PB038, and the percentage of free eczema or DXD released after enzyme cleavage was measured at different time points. The results are shown in Figure 19. Under the conditions of cleavage using cathepsin L, the enzyme cleavage rate of ADC2-PB038 was significantly faster than that of ADC2-A. It was also found that ADC-deruxtecan, ADC1-deruxtecan, and ADC2-deruxtecan were not cleaved by cathepsin B, but were mainly cleaved by cathepsin L.

[0628] It is particularly noteworthy that, as shown in Figure 17, ADC-B exhibits the fastest enzymatic cleavage rate, presumably due to the introduction of three linear glycosyl groups from the side chain of the corresponding linker toxin LDB. However, ADC-A demonstrated better efficacy than ADC-B in the BXPC-3 model. Similarly, PB038's ADC exhibits excellent hydrophilicity, resulting in a significantly faster enzymatic cleavage rate than LDA's ADC. However, the in vivo efficacy of PB038's ADC, especially in models with low to moderate expression, is significantly weaker than that of LDA's ADC. This result suggests that the final drug efficacy is not linearly related to the cleavage rate. ADCs with slower enzymatic cleavage rates may possess a better ability to shield against hydrophobic toxins such as eczema and MMAE, thereby exhibiting better in vivo biological activity.

[0629] In summary, the modular design of the linker allows for regulation of the release rate of toxins from enzyme cleavage, thereby helping to control the efficacy and toxicity of ADCs in vivo.

[0630] Example 41. Plasma stability test of ADC

[0631] Methods: 50 μL of ADC-A solution was diluted with 20 mM histidine solution (pH 5.5), and four aliquots were prepared for each sample. 950 μL of human plasma, monkey plasma, rat plasma, and mouse plasma were added to each sample, respectively. After mixing, the aliquots were pipetted into five equal portions, sealed with sealing film, and placed in a 37°C water bath. The stability of ADC-A in human plasma, monkey plasma, rat plasma, and mouse plasma was investigated at 0, 3, 7, 14, and 21 days. After precipitation with acetonitrile, the free eczema in the ten samples was detected by LC-MS and converted to the percentage of release (Figure 20). The results showed that ADC-A exhibited excellent stability in various plasmas. After 21 days, except for mouse plasma where the eczema release percentage was 1.25%, the release in other plasmas was less than 1%.

[0632] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, Ab-(LD). f Formula (I) in, f is an integer or decimal number ≥ 1, preferably 1.0 to 16.0, more preferably 2.0 to 8.0, and most preferably 4.0 to 8.0; Ab represents an antibody or antigen-binding fragment against ADAM-9 that binds to the target ADAM-9. D is a small molecule drug, preferably a hydrophobic small molecule drug, more preferably a cytotoxic small molecule drug and / or a drug for treating autoimmune diseases and anti-inflammation; L is a linker group that connects the antibody and D, and its structure is -L1-L2-L3-; Wherein, L1 is the linking group used to connect Ab, and L1 is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; This indicates a double or single bond; * indicates the position where L1 connects to Ab. This is the location where L1 and L2 are connected; L2 is a linking group used to connect L1 and L3. Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3; Each X 3 Independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds); Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides; X5 is C(O) or NH; L3 is a linking group for connecting D, and the L3 is selected from the group consisting of AA, self-fractured structural fragments, or AA-self-fractured structural fragments. Among them, AA is a dipeptide, tripeptide or tetrapeptide fragment (i.e. a fragment formed by 2, 3 or 4 amino acids linked by peptide bonds); The fragments that break down are selected from the following group: PAB (p-aminobenzyloxycarbonyl), )， L4 is selected from the following group: And when the L4 is In this case, X3 is an amino acid side chain; Among them, L d L a and L b Select independently from the following groups: In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5; X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene -CO-, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH-, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5; L c Selected from the following group: y and z are each independently 0, 1, 2, 3 or 4; R3, R4, R5, and R6 are hydrophilic chains, and each of them is independently selected from the following group: Among them, n3, n4, n5 and n6 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyl groups, cyclic glycosyl groups (such as cyclic glucosyl, cyclic mannose or cyclic galactosyl), and linear glycosyl groups (such as linear glucosyl, linear mannose or linear galactosyl).

2. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The L2 is a linking group modified by a moiety with a branched hydrophilic chain; The module with branched hydrophilic chains includes at least one (preferably 2, 3, 4 or 5) branched frameworks and multiple (preferably 2, 3, 4 or 5) hydrophilic chains covalently connected to the branched frameworks. Preferably, the moiety of the branched hydrophilic chain is selected from group L4: in, Branched skeleton, * indicates the position where it connects to L1, and * indicates the position where it connects to the hydrophilic chain; R3, R4, R5, and R6 are hydrophilic chains.

3. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The term D refers to cytotoxic small molecule drugs selected from the following group: DNA damaging agents, microtubule inhibitors; The DNA damaging agent is a topoisomerase inhibitor or a DNA binding agent. Preferably, the topoisomerase inhibitor is from the group consisting of: Exatecan (DX8951), DXD, SN-38, 9-nitrocamptothecin, CPT-11, 10-hydroxycamptothecin, and doxorubicin metabolite PNU-159682. The DNA damaging agent is selected from the group consisting of: pyrrolobenzodiazepines (PBD), Duocarmycin, Calicacin; The microtubule inhibitors mentioned are selected from the following group: maytansine derivatives, eribulin, monomethyl alatatin-E (MMAE), monomethyl alatatin-F (MMAF), monomethyl dolastatin 10 (MMAD), tubulysin derivatives, cryptophycin derivatives, and taltobulin.

4. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The L mentioned d L a and L b Independently X1 is -C 1-8 alkylene-NH- or -C 1-8 alkylene-CO-; The L mentioned c for 5. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, R3, R4, R5, and R6 are each independently selected from the following group: Preferably, R3, R4, R5 and R6 are each independently selected from the following group:

6. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The AA mentioned is selected from the following group: Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Phe-Lys (phenylalanine-lysine), Ala-Ala-Asn (alanine-alanine-asparagine), D-Ala-Phe-Lys (D-alanine-phenylalanine-lysine), Gly-Gly-Phe-Gly (glycine-glycine-phenylalanine-glycine), Ala-Ala-Ala (alanine-alanine-alanine), Val-Lys (valine-lysine).

7. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The L4 mentioned is selected from the following group:

8. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The compound has the structure shown in formula (I-1): The definitions of L2 and L3 are as described in claim 1.

9. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The compound has the structure shown in formula (I-2): The definitions of Ab, f, and L2 are as described in claim 1.

10. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The compounds are selected from the following group: The definitions of Ab and f are as described in claim 1.

11. The compound of claim 1, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt, is characterized in that, The Ab is an anti-ADAM9 antibody or its antigen-binding fragment, which contains a heavy chain variable region and a light chain variable region, wherein, The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NO:7-10 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NO:11-12 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NO:13-16 or any variant thereof. The heavy chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NO:17-19 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NO:20-22 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NO:23-24 or any variant thereof. Preferably, the light chain variable region comprises: (1) LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13, or, (2) LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14, or, (3) LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; or, (4) LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:15; The heavy chain variable region includes: (1) HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23, or, (2) HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22, or, (3) HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21 and HCDR3 shown in SEQ ID NO:24; (4) HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22 and HCDR3 shown in SEQ ID NO:23; Preferably, the anti-ADAM9 antibody or its antigen-binding fragment has any of the features selected from the group consisting of (1)-(16): (1) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (2) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (3) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or, (4) The light chain variable region includes LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or, (5) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (6) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (7) The light chain variable region includes LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or, (8) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or, (9) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (10) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (11) The light chain variable region includes LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or, (12) The light chain variable region includes LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or, (13) The light chain variable region comprises LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (14) The light chain variable region includes LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or (15) The light chain variable region includes LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region includes HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22 and HCDR3 shown in SEQ ID NO:23; Preferably, the anti-ADAM9 antibody or its antigen-binding fragment is selected from any one of murine antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, human antibodies or their antigen-binding fragments, or humanized antibodies or their antigen-binding fragments.

12. The compound of claim 11, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, wherein the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region derived from the human κ chain, λ chain, or a variant thereof; Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region derived from the human κ chain; More preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO:

5.

13. The compound of claim 11, wherein a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, wherein the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region or a variant thereof derived from human IgG1, IgG2, IgG3, or IgG4. Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4; Further preferably, the anti-ADAM9 antibody or its antigen-binding fragment further includes a heavy chain constant region as shown in SEQ ID NO:

6.

14. The compound of claim 11, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, wherein the anti-ADAM9 antibody or its antigen-binding fragment comprises a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the following sequences: SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34; and / or The heavy chain variable regions are selected from the following sequences, or have at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:

36.

15. The compound of claim 11, wherein the pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, wherein the anti-ADAM9 antibody or its antigen-binding fragment comprises: The light chain variable region shown in SEQ ID NO: 25 and the heavy chain variable region shown in SEQ ID NO: 26; The light chain variable region shown in SEQ ID NO: 27 and the heavy chain variable region shown in SEQ ID NO: 28; The light chain variable region shown in SEQ ID NO: 27 and the heavy chain variable region shown in SEQ ID NO: 26; The light chain variable region shown in SEQ ID NO: 29 and the heavy chain variable region shown in SEQ ID NO: 26; The light chain variable region shown in SEQ ID NO: 32 and the heavy chain variable region shown in SEQ ID NO: 33; The light chain variable region shown in SEQ ID NO: 32 and the heavy chain variable region shown in SEQ ID NO: 31; The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 35; or The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 36; Preferably, the anti-ADAM9 antibody or its antigen-binding fragment contains a light chain selected from the sequences shown below, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity with the following sequences: SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46; and / or The heavy chains selected from the following sequences, or heavy chains having at least 80%, 85%, 90%, 95% or 99% identity with the following sequences: SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:48; More preferably, the anti-ADAM9 antibody comprises: (1) The light chain shown in SEQ ID NO: 37 and the heavy chain shown in SEQ ID NO: 38; (2) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 40; (3) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 38; (4) The light chain shown in SEQ ID NO: 41 and the heavy chain shown in SEQ ID NO: 38; (6) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 45; (7) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 43; (8) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 47; or (9) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO:

48.

16. A compound of formula (II), or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, HLD formula (II) D is a small molecule drug, preferably a hydrophobic small molecule drug, more preferably a cytotoxic small molecule drug and / or a drug for treating autoimmune diseases and anti-inflammation; L is -L1-L2-L3-; L1 is selected from the following group: in, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; This indicates a double or single bond; * indicates the position where L1 connects to H. This is the location where L1 and L2 are connected; L2 is Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3; Each X3 is independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds); Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides; X5 is C(O) or NH; L4 is selected from the following group: And when the L4 is In this case, X3 is an amino acid side chain; Among them, L d L a and L b Select independently from the following groups: In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5; X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene -CO-, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH-, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5; L c Selected from the following group: y and z are each independently 0, 1, 2, 3 or 4; R3, R4, R5, and R6 are hydrophilic chains, and each of them is independently selected from the following group: Wherein, n3, n4, n5 and n6 are each an integer from 0 to 20; preferably, they are any integers from 0 to 10. R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, cyclic glycosyl (such as cyclic glucosyl, cyclic mannosyl or cyclic galactosyl), linear glycosyl (such as linear glucosyl, linear mannosyl or linear galactosyl); L3 represents AA, AA-PAB, PAB, AA-other self-fracture structures or other self-fracture structures; in, L3 is a linking group for connecting D, and the L3 is selected from the group consisting of AA, self-fractured structural fragments, or AA-self-fractured structural fragments. AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds). The fragments that break down are selected from the following group: PAB (p-aminobenzyloxycarbonyl), )， 17. The compound of claim 16, or its salt, stereoisomer, solvate, or solvate of the salt, characterized in that, D is a cytotoxic small molecule drug selected from the following group: DNA damage agents, microtubule inhibitors; The DNA damaging agent is a topoisomerase inhibitor or a DNA binding agent. Preferably, the topoisomerase inhibitor is from the group consisting of: Exatecan (DX8951), DXD, SN-38, 9-nitrocamptothecin, CPT-11, 10-hydroxycamptothecin, and doxorubicin metabolite PNU-159682. The DNA damaging agent is selected from the group consisting of: pyrrolobenzodiazepines (PBD), Duocarmycin, Calicacin; The microtubule inhibitors mentioned are selected from the following group: maytansine derivatives, eribulin, monomethyl alistatin-E (MMAE), monomethyl alistatin-F (MMAF), monomethyl dolastatin 10 (MMAD), tubulysin derivatives, cryptophycin derivatives, and taltobulin; More preferably, the D is selected from the group consisting of: More preferably, the D is selected from the group consisting of:

18. The compound of claim 16, or its salt, stereoisomer, solvate, or solvate of the salt, characterized in that, The compound, or its salt, stereoisomer, solvate, or solvate of the salt, is selected from the group consisting of:

19. A linker compound, characterized in that, The compound has the structure shown in formula (III): H-L1-L2-L3-H (Formula (III)) L1 is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; This indicates a double or single bond; * indicates the position where L1 connects to H. This is the location where L1 and L2 are connected; L2 is Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3; Each X3 is independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds); Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides; X5 is C(O) or NH; L4 is selected from the following group: And when the L4 is In this case, X3 is an amino acid side chain; Among them, L d L a and L b Select independently from the following groups: In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5; X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene-CO, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH, - Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5; L c Selected from the following group: y and z are each independently 0, 1, 2, 3 or 4; R3, R4, R5, and R6 are hydrophilic chains, and each of them is independently selected from the following group: Wherein, n3, n4, n5 and n6 are each an integer from 0 to 20; preferably, they are any integers from 0 to 10. R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl, cyclic glycosyl (such as cyclic glucosyl, cyclic mannosyl or cyclic galactosyl), linear glycosyl (such as linear glucosyl, linear mannosyl or linear galactosyl); L3 is an AA-self-fractured structural segment, or an AA-self-fractured structural segment; AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds). The fragments selected from the following group: PAB (p-aminobenzyloxycarbonyl), )、 20. An intermediate for preparing a linker compound, characterized in that, The compound has the structure shown in formula (IV): R1'-L2'-L3-D (Formula (IV)) R1' is selected from the following group: -NHFmoc, -NH2 L2' is Among them, m, n, s, t, k and p are each independently 0, 1, 2, 3, 4, 5, 6 or 7; This refers to the position where L2 connects to L1 or L3; Each X3 is independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds); Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides; X5 is C(O) or NH; L4 is selected from the following group: And when the L4 is In this case, X3 is an amino acid side chain; Among them, L d L a and L b Select independently from the following groups: In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5; X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene-CO, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene -NH(=NH)-NH-, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5; L c Selected from the following group: y and z are each independently 0, 1, 2, 3 or 4; R3, R4, R5, and R6 are each independently selected from the following groups: H, Boc, Fmoc, Ac; Wherein, n3, n4, n5 and n6 are each an integer from 0 to 20; preferably, they are any integers from 0 to 10. R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyl groups, cyclic glycosyl groups (such as cyclic glucosyl, cyclic mannosyl, or cyclic galactosyl), linear glycosyl groups (such as linear glucosyl, linear mannosyl, or linear galactosyl), -OAc; L3 is an AA-self-fractured structural segment, or an AA-self-fractured structural segment; AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds). The fragments selected from the following group: PAB (p-aminobenzyloxycarbonyl), )、 21. The intermediate as described in claim 20, characterized in that, The intermediate compound is selected from the group consisting of:

22. An intermediate for preparing a linker compound, characterized in that, The compound has the structure shown in formula (V-1), (V-2), or (V-3): R1 is selected from the following group: hydroxyl, L1' is selected from the following group: Among them, R2 is selected from the following groups: H, C 1-4 Alkyl, C 1-4 Haloalkyl, C 3-6 cycloalkyl groups, 4-7 membered heterocyclic groups; It can be a double bond or a single bond; m, n, s, t, k, and p are each independently 0, 1, 2, 3, 4, 5, 6, or 7; Each X 3 Independently selected from the following groups: amino acid side chain, -NH-C(O)-, -CH2CH2O-, -CH2-, amino acid residue, dipeptide, tripeptide, tetrapeptide (i.e., fragments formed by 2, 3, or 4 amino acids linked by peptide bonds); Each X4 is independently selected from the following groups: amino acid residues, dipeptides, tripeptides, and tetrapeptides; X5 is C(O) or NH; L3' is selected from the following group: AA, Ra, or AA-benzylbenzene compounds; AA can be a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment formed by 2, 3, or 4 amino acids linked together by peptide bonds). Ra is selected from the following group: Among them, L d '、L a 'and L b 'Select independently from the following group:' In this context, u, v, and w are each independently 0, 1, 2, 3, 4, or 5; X1 and X2 are each independently selected from the following group: key, -C 1-8 alkylene-, -C 1-8 Alkylene -NH-, -C 1-8 Alkylene -CO-, -C 1-8 Alkylene -CONH, -C 1-8 Alkylene-NH(=NH)-NH, Where n1 and n2 are each independently 0, 1, 2, 3, 4 or 5; L c Selected from the following group: y and z are each independently 0, 1, 2, 3 or 4; R3', R4', R5', and R6' are each independently selected from the following groups: H, Boc, Fmoc, Ac, Among them, n3, n4, n5 and n6 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; R7 is selected from the following group: C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyl groups, cyclic glycosyl groups (such as cyclic glucosyl, cyclic mannose or cyclic galactosyl), and linear glycosyl groups (such as linear glucosyl, linear mannose or linear galactosyl).

23. The intermediate as described in claim 22, characterized in that, The intermediate compound is selected from the group consisting of:

24. A pharmaceutical composition comprising a compound of Formula I as claimed in any one of claims 1-15, a compound of Formula II as claimed in any one of claims 16-18, or a compound of Formula III as claimed in claim 19, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable diluents, carriers, and / or excipients.

25. A pharmaceutical formulation comprising a compound of Formula I as claimed in any one of claims 1-15, a compound of Formula II as claimed in any one of claims 16-18, or a compound of Formula III as claimed in claim 19, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable diluents, carriers, and / or excipients.

26. Use of a substance X in the preparation of a medicament for the prevention or treatment of cancer or inflammation; in, The substance X is a compound of formula I as described in any one of claims 1-15, a compound of formula II as described in any one of claims 16-18, or a compound comprising the structure shown in formula III as described in claim 19, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 26, or a pharmaceutical formulation as described in claim 25; Preferably, the cancer is a solid tumor or a non-solid tumor; More preferably, the cancer is selected from the group consisting of: esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal carcinoma, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

27. Use of a substance X in the preparation of a medicament for the prevention or treatment of diseases associated with abnormal activity or expression of ADAM-9; in, The substance X is a compound of formula I as described in any one of claims 1-15, a compound of formula II as described in any one of claims 16-18, or a compound comprising the structure shown in formula III as described in claim 19, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 20, or a pharmaceutical formulation as described in claim 25; Preferably, the disease associated with abnormal cell activity is cancer; More preferably, the cancer is a solid tumor or a non-solid tumor; More preferably, the cancer is selected from the group consisting of: esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal carcinoma, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

28. The use of substance X in the preparation of pharmaceuticals; in, The substance X is a compound of formula I as described in any one of claims 1-15, a compound of formula II as described in any one of claims 16-18, or a compound comprising the structure shown in formula III as described in claim 19, or a pharmaceutically acceptable salt, stereoisomer, solvate, or solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 20, or a pharmaceutical formulation as described in claim 25; The drug is used to treat diseases associated with ADAM-9; Preferably, the drug is used to treat cancer, a disease associated with target A; More preferably, the cancer is selected from the group consisting of: esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, nasopharyngeal carcinoma, skin cancer, multiple myeloma, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer; more preferably selected from the group consisting of: pancreatic cancer, colorectal cancer, non-small cell lung cancer, bile duct cancer, gastric cancer.

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