Fused ring compound and antibody-drug conjugate thereof, and preparation method therefor and use thereof

Abstract

A fused ring compound and an antibody-drug conjugate thereof, and a preparation method therefor and the use thereof. The antibody-drug conjugate has a structure as shown in formula (I), and the drug is selected from a topoisomerase inhibitor, a DNA damaging agent, a microtubule inhibitor, an RNA polymerase inhibitor and an anti-metabolite. The prepared antibody-drug conjugate has a superior drug-to-antibody ratio, and has a targeted killing effect on breast cancer, gastric cancer, lung cancer and colorectal cancer.

Description

Fused ring compounds and their antibody-drug conjugates, preparation methods and uses

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application number 202411989455.0, filed on December 31, 2024, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application belongs to the pharmaceutical field and relates to fused ring compounds and their antibody-drug conjugates, preparation methods and uses. Background Technology

[0004] Significant progress has been made in the development of antitumor drugs and targeted tumor therapy using biopharmaceuticals (such as therapeutic antibodies or antibody fragments) and targeted small molecule ligands. However, while biopharmaceuticals are highly targeted, their therapeutic effects on solid tumors are limited; and while bioactive molecules have high killing power against cancer cells, they often lack targeting and frequently damage normal cells, leading to serious toxic side effects.

[0005] Recent studies have found that therapeutic antibodies can be linked to bioactive molecules to form antibody-drug conjugates (ADCs). ADCs combine the targeting properties of antibodies with the activity of bioactive molecules. Antibodies guide ADCs to bind to target cells, where they are subsequently internalized, releasing the drug to kill cells and treat the disease. Because antibodies are specific and targeted to tumor cell-related targets, their application value lies not only in treatment but also in serving as ideal carriers for targeted drug delivery, reducing drug side effects.

[0006] PNU-159682 is an anthracycline compound whose derivatives have shown cytotoxic effects against various tumor cell lines. Due to its excellent cytotoxicity, it is considered a potent antibody-drug conjugate (ADC) cytotoxic agent. Given the growing clinical demand, there is a need to develop safe and effective novel PNU-type antibody-drug conjugates. Summary of the Invention

[0007] This application provides a novel drug linker and its antibody-drug conjugate, which have good antitumor effects, safety and pharmacokinetic properties.

[0008] Drug linker

[0009] On the one hand, this application provides a drug linker, wherein the drug linker is a compound with the structure shown in Formula I or a drug-acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotope-labeled compound, metabolite, or prodrug:

[0010] Formula (I)

[0011] in:

[0012] Q is the structure before it is linked to the antibody or antigen-binding fragment;

[0013] L is the connector sub-part;

[0014] E is the self-eliminating part;

[0015] D represents the bioactive molecule portion;

[0016] n is selected from 1-10.

[0017] In some embodiments, the bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects.

[0018] In some embodiments, the bioactive molecules are each independently selected from cytotoxic compounds or antimetabolites.

[0019] In some embodiments, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor.

[0020] In some embodiments, the topoisomerase inhibitor is selected from camptothecin compounds, anthracycline compounds, etoposide compounds, and PNU compounds.

[0021] In some embodiments, the topoisomerase inhibitor is a DNA topoisomerase inhibitor.

[0022] In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecins, including but not limited to hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan, etc.) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide).

[0023] In some implementations, Q is selected individually from the following structures each time it appears: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears.

[0024] In some implementations, Q is selected individually from the following structures each time it appears: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears.

[0025] In some implementations, Q is selected individually from the following structures each time it appears:

[0026] In some implementations, Q has the following structure:

[0027] In some implementations, L is selected from a divalent structure composed of one or more substituted or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 5-12 heterocyclic, -N(R')-, carbonyl, -O-, glycosyl, tromethamine, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)) r OCH3)) and Lys(R') rand short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D-Leu-Al). a-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu-Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Ala-Ala-Glu, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:44), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:45), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:46), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:47), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 48), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 49), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 50), EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO, Wherein Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R' represents hydrogen, C 1-6 Alkyl groups, polyhydroxy fragments, glycosyl groups, polyethylene glycol-containing fragments, -(CH2CH2O) r -C 1-6 Alkyl group, -C(=O)-(CH2CH2O) r -C 1-6 Alkyl, polysarcosine, -(C(=O)-CH2N(Me)) r -C 1-6 Alkyl groups, carboxylic acid-containing fragments, tetracarboxylic acid residues and their derivatives, EDTA and its derivatives, or DOTA and its derivatives; r is independently selected from integers from 1 to 20 each time it appears.

[0028] In some implementations, L is selected from the following structures: Each time r appears, it is independently selected from an integer between 1 and 20.

[0029] In some implementations, L is selected from the following structures:

[0030] In some implementations, E is selected from a single bond, -NH-CH2-.

[0031] In some implementations, E is selected from single bonds, -NH-CH2- or

[0032] In some implementations, E is -NH-CH2-.

[0033] In some implementations, n in equation (I) is 1, and -ELQ is selected from the following structures:

[0034] LJ'-1:

[0035] LJ'-2:

[0036] LJ'-3:

[0037] The bioactive molecules disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino (-NH2), and secondary amino (-NR). A H), tertiary amino group (-NR) B R C ), or thiol (-SH), where R A R B R C This refers only to non-hydrogen substituents on N, which can be connected to the rest of the part through chemical reactions.

[0038] In some embodiments, the bioactive molecules are each independently linked to the E via -OH, primary amino, secondary amino, tertiary amino, or -SH groups.

[0039] In some embodiments, the bioactive molecule is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs, wherein the compound has the structure shown in formula (III'):

[0040] in,

[0041] X is selected from chemical bonds, -O-, -S-, and -N(R3)-;

[0042] L1 is selected from single bonds and -C(R4)(R5)-;

[0043] R1 is selected from hydrogen, halogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 Alkyne, cyano, HO-(CH2) m -O-(CH2) m -、H2N-(CH2) m -O-(CH2) m - HS-(CH2) m -O-(CH2) m -、HO-(CH2) m -、H2N-(CH2) m - HS-(CH2) m - aryl, benzyl, heteroaryl and heterocyclic groups, the C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2- 6. Acynyl, aryl, benzyl, heteroaryl, and heterocyclic groups may optionally be selected from one or more halogens, C 1-6 Alkyl, C 1- 6-alkoxy, C 1-6 Substituents include alkylamine, hydroxyl, hydroxyalkyl, amino, and aminoalkyl groups;

[0044] R2 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, haloalkyl, amino, aminealkyl, hydroxyalkyl, HO-(CH2) m -O-(CH2) m - and H2N-(CH2) m -O-(CH2) m -;

[0045] m is independently selected from 0-10 (preferably from 1-10);

[0046] R3 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl;

[0047] R4 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl;

[0048] R5 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; or,

[0049] When X is selected from -N(R3)-, R3 can also form a ring with R1 and the atoms attached to it.

[0050] In some embodiments of formula (III'), when R2 is methyl, X is NH, and L is -CH2-, R1 is not H2N-(CH2)2-.

[0051] In some embodiments, the bioactive molecule is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope labels, metabolites, or prodrugs, wherein the compound has the structure shown in formula (III):

[0052] in,

[0053] X is selected from chemical bonds, -O-, -S-, and -N(R3)-;

[0054] R1 is selected from hydrogen, halogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 Alkyne, cyano, HO-(CH2) m -O-(CH2) m -、H2N-(CH2) m -O-(CH2) m -、HO-(CH2) m -、H2N-(CH2) m - aryl, benzyl, heteroaryl and heterocyclic groups, the C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 The alkynyl, aryl, benzyl, heteroaryl, and heterocyclic groups are each optionally composed of one or more groups selected from halogens, C, and D. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Substituents include alkylamine, hydroxyl, hydroxyalkyl, amino, and aminoalkyl groups;

[0055] R2 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, haloalkyl, amino, aminealkyl, hydroxyalkyl, HO-(CH2) m -O-(CH2) m - and H2N-(CH2) m -O-(CH2) m -;

[0056] m is independently selected from 0-10 (preferably from 1-10);

[0057] R3 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; or,

[0058] When X is selected from -N(R3)-, R3 can also form a ring with R1 and the atoms attached to it.

[0059] In some implementations, X is selected from -S- and -N(R3)-.

[0060] In some implementations, X is selected from -S- and -NH-.

[0061] In some implementations, X is selected from chemical bonds, -S- and -N(R3)-.

[0062] In some implementations, X is selected from chemical bonds, -S-, and -NH-.

[0063] In some implementations, R1 is selected from HO-(CH2). m - and H2N-(CH2) m -

[0064] In some implementations, R1 is selected from HO-, HO-CH2- and H2N-CH2-.

[0065] In some implementations, R1 is selected from HO-CH2- and H2N-CH2-.

[0066] In some implementations, R2 is selected from C 1-6 alkyl.

[0067] In some implementations, R2 is methyl.

[0068] In some embodiments, the bioactive molecule is selected from the following compounds:

[0069] In some implementations, D is selected from the following structures:

[0070] In some embodiments, the bioactive molecule is as described in PCT / CN2025 / 131079, the entire contents of which are incorporated herein by reference.

[0071] In some embodiments, the "drug linker" is selected from the following structures:

[0072] J-1:

[0073] J-2:

[0074] J-3:

[0075] Antibody-drug conjugates

[0076] In another aspect, this application provides an antibody-drug conjugate or a pharmaceutically acceptable salt or stereoisomer thereof, said antibody-drug conjugate having the structure shown in formula (II):

[0077] Where L' is as described in any of the L items above;

[0078] E' is as described in any of the above E items;

[0079] D' is as described in any of the above D items;

[0080] A represents an antibody or its antigen-binding fragment;

[0081] Q' is the structural form of Q covalently linked to an antibody or its antigen-binding fragment as described in any of the above items;

[0082] x is selected from 1 to 10.

[0083] In the antibody-drug conjugate, D' can be conjugated to the antibody or its antigen-binding fragment via a linker.

[0084] In some implementations, Q' is selected from the following structures: In this context, each occurrence of p is independently selected from an integer between 1 and 12.

[0085] In some implementations, Q' is selected from the following structures: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears.

[0086] In some implementations, Q' has the following structure:

[0087] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0088] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0089] (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system:

[0090] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0091] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;

[0092] Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0093] or,

[0094] (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0095] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or,

[0096] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0097] Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0098] or,

[0099] (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system:

[0100] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,

[0101] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0102] Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;

[0103] or,

[0104] (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0105] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or,

[0106] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof;

[0107] Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0108] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0109] (1) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Chothia numbering system:

[0110] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0111] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0112] or,

[0113] (2) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system:

[0114] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:18, CDR-H2 of SEQ ID NO:19, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0115] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:33, CDR-H2 of SEQ ID NO:34, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0116] or,

[0117] (3) The following heavy chain variable regions (VH) and light chain variable regions (VL), where CDR is defined according to the Kabat numbering system:

[0118] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,

[0119] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:26, CDR-H2 of SEQ ID NO:27, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;

[0120] or,

[0121] (4) The following heavy chain variable regions (VH) and light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system:

[0122] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:16, CDR-L2 of SEQ ID NO:17, and CDR-L3 of SEQ ID NO:10; or,

[0123] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:28, CDR-H2 of SEQ ID NO:29, and CDR-H3 of SEQ ID NO:30; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:31, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:25.

[0124] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0125] (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or

[0126] (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4;

[0127] The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0128] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0129] (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or

[0130] (b) VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4.

[0131] In some embodiments, the antibody or its antigen-binding fragment further comprises:

[0132] (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and

[0133] (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0134] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.

[0135] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.

[0136] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 41.

[0137] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.

[0138] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0139] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36.

[0140] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0141] (1) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36;

[0142] (2) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0143] (3) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or

[0144] (4) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.

[0145] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0146] (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38;

[0147] (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40;

[0148] (3) The heavy chain comprising the sequence shown in SEQ ID NO: 42, and the light chain comprising the sequence shown in SEQ ID NO: 38; or

[0149] (4) The heavy chain comprising the sequence shown in SEQ ID NO: 43, and the light chain comprising the sequence shown in SEQ ID NO: 40.

[0150] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or may lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0151] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0152] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0153] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0154] In some embodiments, the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37, 39, 42 or 43, undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0155] In some embodiments, the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or 41 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37, 39, 42 or 43 or a variant thereof, lacks a C-terminal lysine residue.

[0156] In some embodiments, A is selected from an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase family.

[0157] In some implementations, A is selected from trastuzumab, pertuzumab, trastuzumab mutant, pertuzumab mutant, or a biepisode antibody or its antigen-binding fragment constructed from trastuzumab and pertuzumab.

[0158] In some embodiments, the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT / mAb-DB ID) of 80.

[0159] Those skilled in the art will understand that the antibody-drug conjugates described in this application can be prepared in a modular manner. For example, a free form of the "drug-linker" (QLED, where Q is the structural form of Q' before covalently linking it to an antibody or its antigen-binding fragment) described above can be obtained first, and then covalently linked to the antibody or its antigen-binding fragment to obtain the antibody-drug conjugates described in this application. Accordingly, in the free form of the "drug-linker," Q is linked to one or more thiol (-SH) or amino (-NH2) groups on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of structures such as -SO2Me, -Br, or pentafluorophenol) or an addition reaction.

[0160] In some implementation schemes, Selected from the following structures:

[0161] ADC J-1:

[0162] ADC J-2:

[0163] ADC J-3:

[0164] Among them, the -S-) in each antibody-drug conjugate x -Ab represents an antibody or its antigen-binding fragment as described in any of the above descriptions. This indicates the specific connection between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment, where x is selected from 1 to 10.

[0165] In some implementation schemes, the -S-) in each antibody-drug conjugate x -Ab is selected from A as described in any of the above.

[0166] In some implementation schemes, the -S-) in each antibody-drug conjugate x -Ab represents an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

[0167] in, This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment.

[0168] In some implementation schemes, This indicates the specific linkage between the thiol group and the pyrimidine group of cysteine ​​in the antibody or its antigen-binding fragment.

[0169] In some embodiments, the antibody or its antigen-binding fragment is trastuzumab or pertuzumab.

[0170] Connector

[0171] On the other hand, the present invention provides a connector with the structural formula -E'-L'). n -Q'-; wherein E', L', Q', and n are as described in any of the above.

[0172] In some embodiments, E' is used to link with D' as described in any one of the present applications, and Q' is used to link with an antibody or its antigen-binding fragment as described in any one of the present applications.

[0173] In some embodiments, the connector structure is as follows:

[0174] LJ-1:

[0175] LJ-2:

[0176] LJ-3:

[0177] In some embodiments, the present invention provides a connector, the connector having the structural formula -EL). n -Q; wherein E, L, Q and n are as described in any of the above.

[0178] In some embodiments, E is used to connect with D as described in any one of the claims of this application.

[0179] In some embodiments, the connector structure is as follows:

[0180] LJ'-1:

[0181] LJ'-2:

[0182] LJ'-3:

[0183] Synthetic intermediates

[0184] In another aspect, the present invention provides the following intermediate compounds and their salts (e.g., pharmaceutically acceptable salts):

[0185] PG1 is a hydrogen or amino protecting group.

[0186] In some embodiments, the amino protecting group is selected from benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), (trimethylsilyl)ethoxymethyl (SEM), methoxycarbonyl (or eth)oxycarbonyl, optionally protected by C. 1-6 Alkyl-substituted triphenylmethyl or tert-butylsulfinyl; preferably, the amino protecting group is selected from benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), (trimethylsilyl)ethoxymethyl (SEM), methoxycarbonyl (or eth)oxycarbonyl, or tert-butylsulfinyl; preferably, the amino protecting group is methoxycarbonyl (Fmoc).

[0187] In some implementations, the intermediate has the following structure:

[0188] All technical features disclosed in this specification, except for mutually exclusive features, can be combined in any way. This invention covers compounds, conjugates, linkers, and intermediates obtained by arbitrary combinations of various embodiments. The compounds, conjugates, linkers, and intermediates of this invention can be optionally substituted with suitable substituents at suitable substitution positions. In some embodiments, the suitable substituents may be selected, for example, from: deuterium, halogen, hydroxyl, cyano, amino, C... 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, C 1-6 alkylamine group, C 3-8 Cycloalkyl, 3-8 membered heterocyclic groups, C 6-10 Aryl and 5-10 heteroaryl compounds.

[0189] Composition

[0190] On the other hand, this application provides compositions of antibody-drug conjugates (ADCs) as described herein. Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC contains a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the compositions are characterized by a drug-antibody ratio (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.

[0191] For example, in any embodiment, the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.

[0192] In some embodiments, the DAR of the ADC compositions described herein is about 1 to 8, for example, about 1.0 to 1.5, about 1.5 to 2.0, about 2.0 to 2.5, about 2.5 to 3.0, about 3.0 to 3.5, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 3.5 to 7.0, about 3.5 to 7.5, about 3.5 to 8.0, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 7. 5, approximately 4.0 to 8.0, approximately 4.5 to 5.0, approximately 4.5 to 5.5, approximately 4.5 to 6.0, approximately 4.5 to 6.5, approximately 4.5 to 7.0, approximately 4.5 to 7.5, approximately 4.5 to 8.0, approximately 5.0 to 5.5, approximately 5.5 to 6.0, approximately 5.5 to 6.5, approximately 5.5 to 7.0, approximately 5.5 to 7.5, approximately 5.5 to 8.0, approximately 6.0 to 6.5, approximately 6.0 to 7.0, approximately 6.0 to 7.5, approximately 6.0 to 8.0, approximately 6.5 to 7.0, approximately 6.5 to 7.5, approximately 6.5 to 8.0, approximately 7.0 to 7.5, approximately 7.0 to 8.0, approximately 7.5 to 8.0.

[0193] In some embodiments, the DAR of the ADC composition described herein is about 4.0 to 8.0.

[0194] In some embodiments, the DAR of the ADC composition described herein is about 6.0 to 8.0.

[0195] In some embodiments, the DAR of the ADC composition described herein is about 7.0 to 7.5.

[0196] In some embodiments, the DAR of the ADC composition described herein is about 3.0 to 6.5.

[0197] Pharmaceutical Composition

[0198] In another aspect, this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates described in any of the preceding claims or a pharmaceutically acceptable salt or stereoisomer thereof, and one or more pharmaceutical excipients.

[0199] The above-described pharmaceutical compositions can act systemically and / or locally, which can be achieved through suitable dosage forms. These dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0200] In some implementations, the antibody-drug conjugates described herein are formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody-drug conjugates of desired purity are mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer in the form of a lyophilized or solution form (Remington's Pharmaceutical Sciences (1980) 16). th (ed., Osol, A.Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates may be administered via any route appropriate for the individual to be treated.

[0201] The pharmaceutical composition described above may contain 0.01 mg to 1000 mg of at least one of the compounds disclosed herein or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof.

[0202] In some embodiments, the pharmaceutical composition may also include one or more other therapeutic agents.

[0203] Another aspect of this disclosure provides a kit comprising a compound of the present disclosure or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition thereof; and optionally, instructions for use of a compound of the present disclosure or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, or an antibody-drug conjugate of the present disclosure or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition thereof.

[0204] application

[0205] The antibody-drug conjugates, drug-linkers, ADC compositions, or drug compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0206] Therefore, this application provides the use of any of the antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of medicaments, particularly in the preparation of medicaments for treating Her2-expressing cancers.

[0207] In addition, this application provides any of the antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the thereof described above for the treatment of diseases or conditions, such as Her2-expressing cancer.

[0208] Additionally, this application provides a method for treating a disease or condition, such as Her2-expressing cancer, comprising the step of administering an effective amount of any of the preceding antibody-drug conjugates, drug-linkers, ADC compositions, or pharmaceutical compositions containing the conjugates to a subject in need.

[0209] In some embodiments, the antibody-drug conjugate, drug-linker, ADC composition, or drug composition is sufficient (e.g., in a subject):

[0210] (1) Inhibits the proliferation of cells (such as tumor cells);

[0211] (2) Inhibits tumor growth;

[0212] (3) Inducing and / or increasing antibody-dependent cytotoxic activity;

[0213] (4) Inhibit HER2-mediated signal transduction;

[0214] (5) Prevention and / or treatment of HER2-mediated diseases / disorders; or

[0215] (6) Any combination of (1)-(5) above.

[0216] In some implementations, the cancer or tumor is selected from solid tumors or hematologic malignancies, such as colon cancer, stomach cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

[0217] definition

[0218] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques obvious to those skilled in the art. Furthermore, laboratory procedures used herein, such as those related to genomics, nucleic acid chemistry, and molecular biology, are standard procedures widely used in their respective fields. While it is believed that the following terms will be readily understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0219] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The amino acid distribution in each region or domain can follow various numbering systems known in the art.

[0220] The term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0221] In this invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.

[0222] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0223] The term "antigen-binding fragment" in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety". See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0224] The term "Fd" refers to an antibody fragment composed of VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).

[0225] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0226] The term "Fc" refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0227] The term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:51) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:52) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some implementations, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, domains containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH of scFv.

[0228] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.

[0229] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0230] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0231] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0232] The term "mouse antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for mouse hybrid fusion cells that can proliferate indefinitely and secrete antibodies, and then screening, preparing and purifying the antibodies; or it refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells after the antigen enters the mouse body.

[0233] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to enhance the immune response. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the intended properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and / or ability to enhance the immune response).

[0234] The term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0235] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0236] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0237] The terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0238] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20Alkyl", C 1- 10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3 Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0239] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

[0240] Information about the sequences involved in this invention is described in the table below:

[0241] The abbreviations used in this article have the following meanings:

[0242] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0243] Nuclear magnetic resonance (¹H NMR) measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0244] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0245] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0246] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0247] I. Preparation of intermediates

[0248] Example 1: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-1)

[0249] Step 1: Preparation of glycyl-glycyl-L-phenylalanine (INT-1-2)

[0250] ((benzyloxy)carbonyl)glycylglycyl-L-phenylalanine (200 mg, 483.77 μmol) was dissolved in methanol (8 mL). After purging with nitrogen, palladium on carbon (20 mg) was added. The gas was purged three times with hydrogen, and the reaction was stirred at room temperature for 3 hours. After the reaction was complete, palladium on carbon was removed by filtration. The filtrate was then concentrated to remove the solvent, giving the title compound (135 mg, 483.37 μmol).

[0251] Its structural characterization data are as follows:

[0252] MS m / z (ESI): 280.2 [M+H] +

[0253] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (INT-1)

[0254] Glycylglycyl-L-phenylalanine (130 mg, 465.46 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetate (170.06 mg, 465.46 μmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (120.32 mg, 930.93 μmol) was added. The reaction was stirred at room temperature for 1 hour. After removing most of the solvent, the reaction solution was purified by rapid column chromatography (C18, water / acetonitrile = 2 / 1) and lyophilized to give the title compound (60 mg, 0.11 mmol).

[0255] Its structural characterization data are as follows:

[0256] MS m / z (ESI): 530.3 [M+H] +

[0257] Example 2 of intermediate preparation: Preparation of (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0258] Step 1: Preparation of L-alanyl-L-alanine (INT-2-2)

[0259] (tert-butoxycarbonyl)-L-alanyl-L-alanine (100 mg, 384.19 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove the solvent, yielding the title compound (160 mg, 370.93 μmol).

[0260] Its structural characterization data are as follows:

[0261] MS m / z (ESI): 161.1 [M+H] +

[0262] Step 2: Preparation of (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (INT-2)

[0263] L-alanyl-L-alanine (160 mg, 412.14 μmol) and 2,5-dioxopyrrolidone-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester (150.58 mg, 412.14 μmol) were dissolved in N,N-dimethylacetamide (2 mL), and N,N-diisopropylethylamine (266.33 mg, 2.06 mmol) was added. The reaction mixture was reacted at 20 °C for 1 hour. The reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and then freeze-dried to give the title compound (125 mg, 304.55 μmol).

[0264] Its structural characterization data are as follows:

[0265] MS m / z (ESI): 428.1 [M+H2O] +

[0266] Example 3 of intermediate preparation: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-3)

[0267] Step 1: N 6 Preparation of 1-(diphenyl(p-tolyl)methyl)-L-lysine (INT-3-2)

[0268] N 2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (10 g, 16.01 mmol) was dissolved in DMF (100 mL), and diethylamine (10 g, 16.01 mmol) was added. The reaction was carried out at 20 °C for 1 hour. After the reaction was completed, the reaction solution was diluted with water and extracted with ethyl acetate. The combined organic phases were dried and concentrated under reduced pressure to remove the solvent, giving the title compound (6.44 g, 14.16 mmol).

[0269] Its structural characterization data are as follows:

[0270] MS m / z(ESI): 403 [M+H] +

[0271] Step Two: N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6 Preparation of -(diphenyl(p-tolyl)methyl)-L-lysine (INT-3-3)

[0272] N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (2 g, 4.97 mmol) and 2,5-dioxopyrrolidone-1-yl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine ester (2.39 g, 5.47 mmol) were dissolved in N,N-dimethylacetamide (20 mL), and N,N-diisopropylethylamine (1.28 g, 9.94 mmol) was added. The reaction mixture was reacted at 20 °C for 2 hours. The reaction solution was subjected to rapid column chromatography (dichloromethane / methanol = 10 / 1) and then evaporated to dryness to give the title compound (2.4 g, 3.23 mmol).

[0273] Its structural characterization data are as follows:

[0274] MS m / z(ESI): 724 [M+H] +

[0275] Step 3: N 2 -(L-valine)-N 6 Preparation of -(diphenyl(p-tolyl)methyl)-L-lysine (INT-3-4)

[0276] N 2 -((((9H-fluoren-9-yl)methoxy)carbonyl)-L-valine)-N 6-(diphenyl(p-tolyl)methyl)-L-lysine (2.4 g, 3.32 mmol) was dissolved in N,N-dimethylacetamide (20 mL), and diethylamine (484.95 mg, 6.63 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was subjected to rapid column chromatography (C18, water / acetonitrile = 2 / 1) and evaporated to dryness to give the title compound (1.98 g, 3.32 mmol).

[0277] Its structural characterization data are as follows:

[0278] MS m / z(ESI): 502 [M+H] +

[0279] Step 4: N 6 -(diphenyl(p-tolyl)methyl)-N 2 Preparation of -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (INT-3)

[0280] N 2 -(L-valine)-N 6 -(diphenyl(p-tolyl)methyl)-L-lysine (1.98 g, 3.25 mmol), 2,5-dioxopyrrolidone-1-yl6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetyl ester, and N,N-dimethylacetamide (20 mL) were dissolved in the solution, and DIPEA (1.26 g, 9.76 mmol) was added. The reaction was carried out at 20 °C for 2 hours. After the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, and the combined organic phases were dried and then evaporated to dryness to give the title compound (2.4 g, 3.03 mmol).

[0281] Its structural characterization data are as follows:

[0282] MS m / z(ESI): 752 [M+H] +

[0283] Example 4 of intermediate preparation: Preparation of (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocyanate fluoromethyl ester (INT-4)

[0284] Step 1: Preparation of 2,5-dioxopyrrolidine-1-yl(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid ester (Int-4-2)

[0285] 1-Hydroxypyrrolidine-2,5-dione (110.03 mg, 956.04 μmol) and (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid (200 mg, 318.68 μmol) were dissolved in dichloromethane (12 mL), and EDCI (183.27 mg, 956.04 μmol) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was diluted with dichloromethane, then washed twice with water, and the organic phase was dried with anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product of the title compound (330 mg, 273.23 μmol, 60% purity).

[0286] Its structural characterization data are as follows:

[0287] MS m / z (ESI): 725.2 [M+H] +

[0288] Step 2: Preparation of (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzo-2-thiocarboxylic acid (Int-4)

[0289] 2,5-dioxopyrrolidine-1-yl(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-carboxylic acid ester (330 mg, 273.23 μmol, 60% purity) was dissolved in DMF (7 mL), and a solution of sodium hydrosulfide (30.64 mg, 546.46 μmol) in DMF (7 mL) was added dropwise. The system darkened in color, and the reaction was stirred at room temperature for 0.5 hours. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (50 mg, 77.68 μmol).

[0290] Its structural characterization data are as follows:

[0291] MS m / z (ESI): 644.2 [M+H] +

[0292] Its preparation method is as follows:

[0293] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0294] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0295] II. Compound Preparation Examples

[0296] Example 1: Preparation of S-((S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentazatrisane-22-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1)

[0297] Step 1: Preparation of (9H-fluorene-9-yl)methyl(2-(((2-iodoethoxy)methyl)amino)-2-oxoethyl)carbamate (J-1-2)

[0298] (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (500 mg, 1.36 mmol) was dissolved in THF (5.00 mL), followed by the addition of 2-iodoethanol (479.62 mg, 2.79 mmol), and then p-toluenesulfonic acid monohydrate (51.64 mg, 271.46 μmol). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was dissolved in dichloromethane and purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%), followed by further concentration under reduced pressure to obtain the title compound (400 mg, 832.82 μmol).

[0299] Its structural characterization data are as follows:

[0300] MS m / z (ESI): 498.1 [M+H2O] +

[0301] Step 2: Preparation of S-(1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1-3)

[0302] Directly to the theoretically contained (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene The reaction mixture of 2-thiocarboxylic acid (78.35 mg, 163.13 μmol), excess sodium hydrosulfide (16 mg, 285.39 μmol), and DMF (3 mL) from the previous step was reacted with (9H-fluorene-9-yl)methyl(2-(((2-iodoethoxy)methyl)amino)-2-oxoethyl)carbamate (78.35 mg, 163.13 μmol). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%) and concentrated again under reduced pressure to give the title compound (70 mg, 70.28 μmol).

[0303] Its structural characterization data are as follows:

[0304] MS m / z (ESI): 996.2 [M+H] + ; 1019.2 [M+Na] +

[0305] Step 3: Preparation of S-(2-((2-aminoacetamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1-4)

[0306] S-(1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (20 mg, 20.08 μmol) was dissolved in DMF (1 mL), and piperidine (17.10 mg, 200.80 μmol) was added dropwise. The system turned black instantly, and the mixture was stirred at room temperature for 15 minutes. After the reaction was completed, the reaction solution was diluted with ethyl acetate and washed with plenty of water, followed by washing with organic phase brine, drying with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the crude product of the title compound (15 mg, 19.38 μmol), which was used directly in the next reaction without purification.

[0307] Its structural characterization data are as follows:

[0308] MS m / z (ESI): 774.3 [M+H] +

[0309] Step 4: Preparation of S-((S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxa-5,8,11,14,17-pentazatrisane-22-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-1)

[0310] S-(2-((2-aminoacetamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene 2-Thiocarboxylic acid ester (15 mg, 19.38 μmol) and (6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)glycylglycyl-L-phenylalanine (10.27 mg, 19.38 μmol) were dissolved in DMF (2 mL), DIPEA (5.01 mg, 38.77 μmol) was added, followed by PyBOP (12.11 mg, 23.26 μmol), and the mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (3.63 mg, 2.77 μmol, 98% purity).

[0311] Its structural characterization data are as follows:

[0312] MS m / z (ESI): 1286.4 [M+H] +

[0313] Its preparation method is as follows:

[0314] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0315] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0316] Example 2: Preparation of S-((7S,10S,13S)-7,10,13-trimethyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-2)

[0317] Step 1: Preparation of (9H-fluorene-9-yl)methyl(S)-(1-(((2-iodoethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (J-2-2)

[0318] (S)-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propamido)methyl ester (1 g, 2.62 mmol) was dissolved in THF (10 mL), followed by the addition of 2-iodoethanol (924.06 mg, 5.37 mmol), and then p-toluenesulfonic acid monohydrate (99.48 mg, 523.00 μmol). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was extracted with water and ethyl acetate, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate = 0-100%) and concentrated again under reduced pressure to give the title compound (928 mg, 1.88 mmol).

[0319] Its structural characterization data are as follows:

[0320] MS m / z (ESI): 512.1 [M+H2O] +

[0321] Step 2: Preparation of S-((S)-1-(9H-fluorene-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-2-3)

[0322] Directly to the theoretically contained (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thio A mixture of substituted carboxylic acid (127 mg, 197.31 μmol), DMF (5 mL), and excess sodium hydrosulfide (28.80 mg, 513.71 μmol) from the previous reaction was added to (9H-fluorene-9-yl)methyl(S)-(1-(((2-iodoethoxy)methyl)amino)-1-oxopropane-2-yl)carbamate (100 mg, 202.30 μmol). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was used directly in the next reaction at the theoretical yield (127 mg, 197.31 μmol) without any treatment.

[0323] Its structural characterization data are as follows:

[0324] MS m / z (ESI): 644.2 [M+H] +

[0325] Step 3: Preparation of S-(2-(((S)-2-aminopropamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thioester (J-2-4)

[0326] S-((S)-1-(9H-fluorene-9-yl)-5-methyl-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4, 5]Oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (40 mg, 27.72 μmol) was dissolved in DMF (2.00 mL), and piperidine (17.24 mg, 202.47 μmol) was added dropwise. The system turned black instantly, and the reaction was stirred at room temperature for 15 minutes. After the reaction was complete, the reaction solution was diluted with ethyl acetate, washed with plenty of water, washed with organic phase brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product of the title compound (20 mg, 25.39 μmol), which was used directly in the next reaction without purification.

[0327] Its structural characterization data are as follows:

[0328] MS m / z(ESI): 788.3 [M+H] +

[0329] Step 4: Preparation of S-((7S,10S,13S)-7,10,13-trimethyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-2)

[0330] (6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-alanyl-L-alanine (10.42 mg, 25.39 μmol) and S-(2-(((S)-2-aminopropionamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4'] The crude product of [3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thioester (20 mg, 25.39 μmol) was dissolved in DMF (2 mL), followed by the addition of DIPEA (16 mg, 123.80 μmol) and then PyBOP (15.85 mg, 30.46 μmol). The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain the title compound (6.65 mg, 5.35 μmol, 95% purity).

[0331] Its structural characterization data are as follows:

[0332] MS m / z (ESI): 1181.4 [M+H] +

[0333] Its preparation method is as follows:

[0334] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0335] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0336] Example 3: Preparation of S-((10S,13S)-10-(4-aminobutyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-3)

[0337] Step 1: S-((10S,13S)-10-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosoe-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy- Preparation of 7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-3-1)

[0338] N 6 -(diphenyl(p-tolyl)methyl)-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine)-L-lysine (29.54 mg, 39.29 μmol) and S-(2-((2-aminoacetamido)methoxy)ethyl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3': The crude product of [4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thioester (38 mg, 49.11 μmol) was mixed with 3 mL of DMF, followed by dropwise addition of DIPEA (19.04 mg, 147.32 μmol), and then BOP condensing agent (21.72 mg, 49.11 μmol). The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate and washed several times with water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane-(dichloromethane:methanol = 3:1) = 0-50%) and concentrated again under reduced pressure to obtain the title compound (43 mg, 28.52 μmol).

[0339] Its structural characterization data are as follows:

[0340] MS m / z (ESI): 1507.5 [M+H] +

[0341] Step 2: Preparation of S-((10S,13S)-10-(4-aminobutyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosico-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester (J-3)

[0342] S-((10S,13S)-10-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-13-isopropyl-20-(2-(methanesulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosoe-19-yn-1-yl)(2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aR,9S) Dichloromethane (2 mL) was added to 43 mg (28.52 μmol) of 9-methoxy-1-methyloctahydro-1H-pyrano[4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrabenzophenanthrene-2-thiocarboxylic acid ester, followed by formic acid (0.2 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure at room temperature to remove the solvent. The crude product was dissolved in acetonitrile, purified by preparative high performance liquid chromatography, and then freeze-dried to obtain the title compound (1.35 mg, 0.99 μmol, 95% purity).

[0343] Its structural characterization data are as follows:

[0344] MS m / z (ESI): 1251.4 [M+H] + 626.4 [M / 2+H] +

[0345] Its preparation method is as follows:

[0346] Column: Waters Xbridge Prep C18 OBD (5μm*19mm*150mm)

[0347] Mobile phase A: Acetonitrile; Mobile phase B: Water (0.05% ammonium bicarbonate)

[0348] Other compounds of the present invention can be prepared by referring to the methods described in the above embodiments.

[0349] III. Examples of Antibody-Drug Conjugate Preparation

[0350] 1. Preparation of Trastuzumab-J-1

[0351] Take 0.608 mL of trastuzumab (22.5 mg / mL), dilute with 30.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 51.9 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 173.5 μL (10 mM) of J-1 dissolved in dimethyl sulfoxide (DMSO) in 18 times the amount of antibody, mix well, and incubate at room temperature for 2 h. After that, replace the buffer solution with 20 mM histidine buffer solution at pH 5.5 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (Trastuzumab-J-1). The DAR value was determined by mass spectrometry to be 3.1.

[0352] 2. Preparation of Trastuzumab-J-2

[0353] Take 0.756 mL of trastuzumab (22.5 mg / mL), dilute with 38.0 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 64.4 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 18 times the amount of J-2 (222.0 μL, 10 mM) dissolved in dimethyl sulfoxide and mix well. Incubate at room temperature for 2 h. After that, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-J-2). The DAR value was determined by mass spectrometry to be 3.0.

[0354] 3. Preparation of Trastuzumab-J-3

[0355] Take 0.629 mL of trastuzumab (22.5 mg / mL), dilute with 31.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60), then adjust the pH to 7.6 with 1 M Na2HPO4 solution. Add 10 mM TCEP (tris(2-carboxyethyl)phosphine, 53.6 μL, pH 7.60) solution and mix well. Incubate at room temperature for 1.5 h. Then add 12 times the amount of J-3 (124.0 μL, 10 mM) solution dissolved in dimethyl sulfoxide, mix well, and incubate at room temperature for 2 h. After that, replace the buffer with 20 mM histidine buffer solution at pH 5.5 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., Trastuzumab-J-3). The DAR value was determined by mass spectrometry to be 6.2.

[0356] Other antibody-drug conjugates of the present invention can also be prepared by conjugation using the above preparation examples.

[0357] IV. Biological Examples

[0358] 1. Evaluate the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model.

[0359] The formulation containing the ADC of the present invention was administered to a subcutaneously transplanted human gastric cancer cell line NCI-N87 mouse CDX model via tail vein injection. Tumor volume and animal weight changes were measured weekly to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[0360] test drug

[0361] Take an appropriate amount of ADC and administer it at a dose of 1 mg / kg, as detailed below. Dilute the stock solution to the administration solution using 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[0362] Laboratory animals and cell lines

[0363] Balb / c Nude mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[0364] Human gastric cancer cells NCI-N87 (ATCC)

[0365] Experimental grouping and evaluation methods

[0366] Select tumors with an average volume of approximately 150 mm. 3Tumor-bearing mice were randomly assigned to groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as solvent control, Vehicle) or ADC, with the dosing frequency as detailed in the specific examples. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0367] The tumor growth inhibition rate (TGI) (%) is calculated using the following formula to evaluate the antitumor efficacy of ADCs: V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1 - (V) T末 -V T0 ) / V T0 ]*100%.

[0368] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0369] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0370] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0371] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0372] The relative tumor proliferation rate (T / C%) is calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs: T / C(%) = (V T末 / V T0 ) / (V C末 / V C0 )*100%.

[0373] (1) Pharmacodynamic assay of anti-human Her2 antibody-drug conjugate in NCI-N87 model

[0374] NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and subcutaneously inoculated into female Balb / c Nude mice to establish a gastric cancer model. The tumors were cultured until the average volume reached approximately 150 mmHg. 3 At approximately 10:00 AM, patients were randomly divided into groups according to tumor size, in the following order: solvent control group (i.e., negative control, Vehicle group), Trastuzumab-J-1 (1 mg / kg), Trastuzumab-J-2 (1 mg / kg), and control DS8201 (1 mg / kg) (Note: DS8201 is an ADC targeting human HER2 developed by Daiichi Sankyo, and the sample used in this experiment was prepared by Kelun Biotech). All groups were administered via tail vein injection (iv) on Day 0, for a total of 1 administration.

[0375] The ADC of this invention significantly inhibited tumor growth in the NCI-N87 gastric cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rate (TGI) of the Trastuzumab-J-1, Trastuzumab-J-2, and DS8201 groups of this invention were 47.31%, 67.17%, and 45.42%, respectively. On Day 21, no animal deaths or significant weight loss were observed in any of the treatment groups, and no obvious drug toxicity was observed. The mice tolerated the ADC of this invention well during the treatment period. Specific results are shown in Table 1.

[0376] Table 1. Human gastric cancer cell NCI-N87 CDX model

[0377] The above results demonstrate that the antibody-drug conjugate of the present invention has significant antitumor effects and good safety.

[0378] 2. Evaluate the inhibitory effect of antibody-drug conjugates on tumor growth in a mouse subcutaneous xenograft model.

[0379] The formulation containing the ADC of the present invention was administered to a subcutaneously transplanted human breast cancer cell JIMT-1 mouse CDX model via tail vein injection. Tumor volume and animal weight changes were measured weekly to calculate the tumor-suppressing efficacy of the ADC of the present invention in tumor-bearing mice.

[0380] test drug

[0381] Take an appropriate amount of ADC and administer once at a dose of 3 mg / kg. Specific dosage details are provided below. Dilute the stock solution to the administration solution using 0.9% NaCl injection. Use 0.9% NaCl injection as a solvent control (Vehicle).

[0382] Laboratory animals and cell lines

[0383] NOD SCID mice (Sichuan Vital River Laboratory Animal Technology Co., Ltd.)

[0384] JIMT-1 human breast cancer cells (Nanjing Kebai)

[0385] Experimental grouping and evaluation methods

[0386] Select tumors with an average volume of approximately 150 mm. 3 Tumor-bearing mice were randomly assigned to groups (the number of groups was determined based on the sample size). Each group was administered either 0.9% NaCl injection (hereinafter referred to as solvent control, Vehicle) or ADC, with the dosing frequency as detailed in the specific examples. The administration method was tail vein injection, with a volume of 10 ml / kg. Tumor diameter was measured weekly using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Animal mortality was observed and recorded daily.

[0387] The tumor growth inhibition rate (TGI) (%) is calculated using the following formula to evaluate the antitumor efficacy of ADCs: V T末 >V T0 TGI(%) = [1 - (V) T末 -V T0 ) / (V C末 -V C0 )]*100% or V T末 ≤V T0 TGI(%) = [1 - (V) T末 -V T0 ) / V T0 ]*100%.

[0388] Where V T末 Mean tumor volume at the end of the experiment in the treatment group;

[0389] V T0 Mean tumor volume at the start of treatment in the treatment group;

[0390] V C末 Mean tumor volume at the end of the experiment in the negative control group;

[0391] V C0 Mean tumor volume at the start of drug administration in the negative control group;

[0392] The relative tumor proliferation rate (T / C%) is calculated using the following formula to evaluate the tumor-suppressive efficacy of ADCs: T / C(%) = (V T末 / V T0 ) / (V C末 / V C0)*100%.

[0393] (1) Pharmacodynamic assay of anti-human Her2 antibody-drug conjugate in JIMT-1 model

[0394] JIMT-1 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. JIMT-1 cells in the exponential growth phase were collected, resuspended in PBS containing 50% matrix gel to a suitable concentration, and subcutaneously inoculated into female NOD SCID mice to establish a breast cancer model. The tumors were cultured until the average volume reached approximately 150 mm². 3 At approximately 10:00 AM, patients were randomly divided into three groups based on tumor size: a solvent control group (i.e., a negative control, Vehicle group), a Trastuzumab-J-2 (3 mg / kg) group, and a Trastuzumab-J-3 (3 mg / kg) group. The drugs were administered on Day 0, and a total of one administration was given.

[0395] The ADC of this invention significantly inhibited tumor growth in the JIMT-1 breast cancer xenograft model. Compared with the Vehicle group, the tumor growth inhibition rate (TGI) of the Trastuzumab-J-2 (3 mg / kg) group and the Trastuzumab-J-3 (3 mg / kg) group were 99.21% and 127.32%, respectively. No animals died in any treatment group on Day 28, and the mice tolerated the ADC of this invention well during the treatment period. Specific results are shown in Table 2.

[0396] Table 2. JIMT-1CDX model of human breast cancer cells.

[0397] The above results demonstrate that the antibody-drug conjugate of the present invention has significant antitumor effects and good safety.

[0398] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, said compound having the structure shown in formula (I): in: Q is the structure before it is linked to the antibody or antigen-binding fragment; L is the connector sub-part; E is the self-eliminating part; D represents the bioactive molecule portion; n is selected from 1-10.

2. The compound of claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, Q is selected independently from the following structures: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears; Preferably, Q is selected from the following structures: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears; Preferably, Q is selected from the following structures:

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, L is a divalent structure composed of one or more of the following substituted or unsubstituted structural segments: C 1-6 Alkylene, 6-10 aryl, 5-6 heteroaryl, 5-12 heterocyclic, -N(R')-, carbonyl, -O-, glycosyl, tromethamine, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH2CH2(OCH2CH2)) r OCH3)) and Lys(R') r and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gly, D- Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Gly-Val-Ala, Glu -Val-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:44), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:45), Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:46), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:47), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 48), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 49), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 50), Ala-Ala-Glu, EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, NOPO, Wherein Ra is EDTA, EGTA, DOTA, NOTA, DEPA, NEPA, PCTA, or NOPO; R' represents hydrogen, C 1-6 Alkyl groups, polyhydroxy fragments, glycosyl groups, polyethylene glycol-containing fragments, -(CH2CH2O) r -C 1-6 Alkyl group, -C(=O)-(CH2CH2O) r -C 1-6 Alkyl, polysarcosine, -(C(=O)-CH2N(Me)) r -C 1-6 Alkyl groups, carboxylic acid-containing fragments, tetracarboxylic acid residues and their derivatives, EDTA and its derivatives, or DOTA and its derivatives; r is independently selected from integers from 1 to 20 each time it appears; Preferably, L is selected from the following structures: Each time r appears, it is independently selected from an integer between 1 and 20.

4. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, E are each independently selected from single bonds, -NH-CH2-, Preferably, E is -NH-CH2-.

5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein, The bioactive molecules are each independently selected from antitumor drugs or compounds with antitumor effects; Preferably, the bioactive molecules are each independently selected from cytotoxic compounds or antimetabolites; Preferably, the cytotoxic compound is a DNA damaging agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, or a microtubule inhibitor; Preferably, the topoisomerase inhibitor is a DNA topoisomerase inhibitor; Preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecins, including but not limited to hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan, etc.) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); Preferably, D is connected to E via -OH, a primary amino group or a secondary amino group, or -SH, respectively; Preferably, the bioactive molecule is selected from compounds of formula (III') or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs. in, X is selected from chemical bonds, -O-, -S-, and -N(R3)-; L1 is selected from single bonds and -C(R4)(R5)-; R1 is selected from hydrogen, halogens, and C. 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 Alkyne, cyano, HO-(CH2) m -O-(CH2) m -、H2N-(CH2) m -O-(CH2) m - HS-(CH2) m -O-(CH2) m -、HO-(CH2) m -、H2N-(CH2) m - HS-(CH2) m - aryl, benzyl, heteroaryl and heterocyclic groups, the C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2- 6. Acynyl, aryl, benzyl, heteroaryl, and heterocyclic groups may optionally be selected from one or more halogens, C 1-6 Alkyl, C 1- 6-alkoxy, C 1-6 Substituents include alkylamine, hydroxyl, hydroxyalkyl, amino, and aminoalkyl groups; R2 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, haloalkyl, amino, aminealkyl, hydroxyalkyl, HO-(CH2) m -O-(CH2) m - and H2N-(CH2) m -O-(CH2) m -; m is independently selected from 0-10 (preferably from 1-10); R3 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; R4 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; R5 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; or, When X is selected from -N(R3)-, R3 can also be linked with R1 and its attached atoms to form a ring; Preferably, the bioactive molecule is selected from compounds of formula (III) or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs. in, X is selected from chemical bonds, -O-, -S-, and -N(R3)-; R1 is selected from hydrogen, halogen, C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 Alkyne, cyano, HO-(CH2) m -O-(CH2) m -、H2N-(CH2) m -O-(CH2) m -、HO-(CH2) m -、H2N-(CH2) m - aryl, benzyl, heteroaryl and heterocyclic groups, the C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl, C 2-6 The alkynyl, aryl, benzyl, heteroaryl, and heterocyclic groups are each optionally composed of one or more groups selected from halogens, C, and D. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Substituents include alkylamine, hydroxyl, hydroxyalkyl, amino, and aminoalkyl groups; R2 is selected from hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, haloalkyl, amino, aminealkyl, hydroxyalkyl, HO-(CH2) m -O-(CH2) m - and H2N-(CH2) m -O-(CH2) m -; m is independently selected from 0-10 (preferably from 1-10); R3 is selected from H and C. 1-6 Alkyl, haloalkyl, and hydroxyl; or, When X is selected from -N(R3)-, R3 can also be linked with R1 and its attached atoms to form a ring; Preferably, the bioactive molecule is selected from the following compounds:

6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is selected from the structures shown below: J-1: J-2: J-3:

7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, wherein the compound is coupled to an antibody or antigen fragment by a substitution reaction (e.g., removal of structures such as -SO2Me, -Br, or pentafluorophenol) or by an addition reaction.

8. An antibody-drug conjugate or a pharmaceutically acceptable salt or stereoisomer thereof, said conjugate having the following structure: in: L' is as described in any one of claims 1-7; E' is as described in any one of claims 1-7; D' is as described in any one of claims 1-7; A represents an antibody or its antigen-binding fragment; Q' is the structural form of Q as described in any one of claims 1-7 after covalently linking it with an antibody or its antigen-binding fragment; x is selected from 1 to 10; Preferably, in the antibody-drug conjugate, D' can be conjugated to the antibody or its antigen-binding fragment via a linker; Preferably, the antibody or its antigen-binding fragment can specifically bind to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.

9. The antibody-drug conjugate of claim 8 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein, Q' is selected from the following structure: Alternatively, Q' can be selected from the following structures: Where R is H or C 1-6 Alkyl group; p is selected independently from integers 1-12 each time it appears; Preferably, Q' is selected from the following structures:

10. The antibody-drug conjugate of claim 8 or 9, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein, The antibody or its antigen-binding fragment comprises: (1) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Chothia numbering system: (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (2) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the AbM numbering system: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence SEQ ID NO:18 or a variant thereof, CDR-H2 with the sequence SEQ ID NO:19 or a variant thereof, and CDR-H3 with the sequence SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence SEQ ID NO:10 or a variant thereof; or, (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:33 or a variant thereof, CDR-H2 with sequence SEQ ID NO:34 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (3) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the Kabat numbering system: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or, (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:26 or a variant thereof, CDR-H2 with sequence SEQ ID NO:27 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:22 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:23 or a variant thereof, CDR-L2 with sequence SEQ ID NO:24 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions; or, (4) The following heavy chain variable regions (VH) and / or light chain variable regions (VL), wherein the CDR is defined according to the IMGT numbering system: (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:13 or a variant thereof, CDR-H2 with sequence SEQ ID NO:14 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:15 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:16 or a variant thereof, CDR-L2 with sequence SEQ ID NO:17 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof; or, (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:28 or a variant thereof, CDR-H2 with sequence SEQ ID NO:29 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:30 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:31 or a variant thereof, CDR-L2 with sequence SEQ ID NO:32 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:25 or a variant thereof; Wherein, the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

11. The antibody-drug conjugate according to any one of claims 8-10, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein, The antibody or its antigen-binding fragment comprises: (a) VH or a variant thereof shown in SEQ ID NO: 1, and / or VL or a variant thereof shown in SEQ ID NO: 2; or (b) VH or a variant thereof shown in SEQ ID NO: 3, and / or VL or a variant thereof shown in SEQ ID NO: 4; The variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with its source sequence, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions; Preferably, the antibody or its antigen-binding fragment comprises: (a) VH shown in SEQ ID NO: 1, and VL shown in SEQ ID NO: 2; or (b) VH shown in SEQ ID NO: 3, and VL shown in SEQ ID NO: 4; Preferably, the antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids). Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region. Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 41; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 41 and a light chain constant region (CL) as shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; (2) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (3) A heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or (4) A heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 41, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36; Preferably, the antibody or its antigen-binding fragment comprises: (1) The heavy chain comprising the sequence shown in SEQ ID NO: 37, and the light chain comprising the sequence shown in SEQ ID NO: 38; (2) The heavy chain comprising the sequence shown in SEQ ID NO: 39, and the light chain comprising the sequence shown in SEQ ID NO: 40; (3) The heavy chain comprising the sequence shown in SEQ ID NO: 42, and the light chain comprising the sequence shown in SEQ ID NO: 38; or (4) The heavy chain comprising the sequence shown in SEQ ID NO: 43, and the light chain comprising the sequence shown in SEQ ID NO:

40.

12. The antibody-drug conjugate or its pharmaceutically acceptable salt or stereoisomer according to any one of claims 8-11, wherein Q' is linked to a thiol group (-SH) or an amino group (-NH2) on A.

13. The antibody-drug conjugate according to any one of claims 8-12, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein, The antibody or its antigen-binding fragment is selected from the antibody or its antigen-binding fragment as described in claim 10 or 11; x is 1 to 8.

14. The antibody-drug conjugate according to any one of claims 8-13, or a pharmaceutically acceptable salt or stereoisomer thereof. Selected from: ADC J-1: ADC J-2: ADC J-3: in, -S- in each antibody-drug conjugate x -Ab represents an antibody or antigen-binding fragment thereof as described in any one of claims 8-13, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO:1 and VL as shown in SEQ ID NO:2; wherein x is 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in, This indicates the specific linkage between the thiol group and the pyrimidine group in the antibody or its antigen-binding fragment.

15. A composition of an antibody-drug conjugate, said composition comprising one or more antibody-drug conjugates according to any one of claims 8-14; preferably, the DAR value (drug-antibody conjugate ratio) of said composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7 ~8, 7~9, 7~10, 8~9, 8~10, or 9~10, preferably 3~6, ​​for example, 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5~6.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 5.0~5.5, 5.0~6.0, 5.5~6.0, 6.0~8.0, 6.5~8.0, 7.0~8.0, 7.5~8.0, or 7.5~8.

5.

16. A pharmaceutical composition comprising the compound of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, the antibody-drug conjugate of any one of claims 8-14 or a pharmaceutically acceptable salt or stereoisomer thereof, or the composition of claim 15, and one or more pharmaceutical excipients.

17. The use of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug of any one of claims 8-14 or a pharmaceutically acceptable salt or stereoisomer of the compound of any one of claims 15 or the composition of claim 16 in the preparation of a medicament for treating cancer; Preferably, the cancer is a cancer that expresses HER2; Preferably, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

18. A method of treating cancer, comprising administering to a subject in need an effective amount of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug, or an antibody-drug conjugate or a pharmaceutically acceptable salt or stereoisomer of any one of claims 8-14, or a composition of claim 15, or a pharmaceutical composition of claim 16; Preferably, the cancer is a cancer that expresses HER2; Preferably, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

19. The compound of any one of claims 1-7 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite or prodrug of the same, or the antibody-drug conjugate of any one of claims 8-14 or a pharmaceutically acceptable salt or stereoisomer of the same, or the composition of claim 15, or the pharmaceutical composition of claim 16, for the treatment of a disease or condition, such as cancer; Preferably, the cancer is a cancer that expresses HER2; Preferably, the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), colon cancer, and lymphoma.

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