Antibody-drug conjugate and use thereof

Abstract

Provided are an antibody-drug conjugate having an anti-tumor effect, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof or an isotopically labeled substance thereof, and the use thereof and a preparation method therefor.

Description

An antibody-drug conjugate and its uses

[0001] This application requires the applicant to have:

[0002] Priority rights to the earlier application filed with the China National Intellectual Property Administration on August 16, 2024, with patent application number 202411135673.8 and title "An antibody-drug conjugate and its use therein";

[0003] The full text of the prior application is incorporated herein by reference. Technical Field

[0004] This invention belongs to the fields of biotechnology and medicine, and specifically relates to an antibody-drug conjugate, its uses, and its preparation method. Background Technology

[0005] Antibody-drug conjugates (ADCs) are currently one of the hottest areas in anti-tumor biopharmaceutical research. To date, 14 ADCs have been marketed. They are: Mylotarg (marketed in 2000) for the treatment of CD33-positive acute myeloid leukemia (AML); Adcetris (marketed in 2011) for Hodgkin's lymphoma; Kadcyla (marketed in 2013) for Her2-positive breast cancer; Besponsa (marketed in 2017) for B-cell lymphoblastic leukemia; Lumoxiti (marketed in 2018) for relapsed or refractory hairy cell leukemia; Polivy (marketed in 2019) for relapsed or refractory diffuse large B-cell lymphoma; and [unclear - possibly related to urothelial pylori treatment]. Padcev (launched in 2019) for cancer; Enhertu (launched in 2019) for metastatic HER2-positive breast cancer; Trodelvy (launched in 2020) for triple-negative breast cancer; Akalux (launched in 2020) for head and neck cancer; Zynlonta (launched in 2021) for large B-cell lymphoma; Aidixi (launched in 2021) for HER2-positive gastric cancer; Tivdak (launched in 2022) for recurrent or metastatic cervical cancer; and Elahere (launched in 2022) for platinum-resistant ovarian cancer. It is worth noting that Mylotarg was withdrawn from the market in 2010 due to its high toxicity, but was reinstated in 2017 after adjustments to its clinical dosage. Meanwhile, Blenrep received accelerated approval for multiple myeloma in 2020, but was withdrawn due to unsatisfactory subsequent clinical results; it has now been approved for reinstatement in Europe.

[0006] The antibody conjugated drug comprises three parts of antibody, linker and toxin, wherein the antibody is closely related to the disease target, the linker and the drug determine the effectiveness and safety of the ADC, and are the most core part. In the ADC drugs that have been listed, most of them adopt the technology of Seattle decades ago, that is, using microtubule inhibitors as active molecules, and using cathepsin hydrolysis linker, and realizing coupling through maleimide and cysteine of the antibody. The ADC obtained by the technology often has larger toxicity, strong hydrophobicity and poor pharmacokinetic properties. Although the ADC obtained by the hydrophilic tetrapeptide linker adopted by Dailchikyo is greatly improved, it still has a large difference from the naked antibody. Although the ADC obtained by the polyethylene glycol containing and salt forming lysine linker adopted by Trodelvy has better hydrophilicity, the stability is poor.

[0007] The dipeptide sequence of the traditional antibody conjugated drug as the enzyme cutting recognition site of the linker can be cut by cathepsin. Cathepsin mainly exists in the lysosome in the cell, but activity can also be observed outside the cell, which poses a risk of non-specific drug release for most antibody conjugated drugs. The antibody conjugated drug involved in the present application exhibits excellent biological activity to the targeted cells, and the improved tripeptide sequence cannot be recognized by cathepsin B, which will reduce the exposure of cytotoxic drugs to normal tissues, which will help to improve the safety and tolerance to treatment. SUMMARY

[0008] The present application provides an antibody drug conjugate having a structure shown in formula (I), and its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotope label: Ab-(Z-W-D) m (I)

[0009] Ab is selected from an antibody or an antigen binding fragment;

[0010] Z is a linker unit;

[0011] D is a drug unit;

[0012] W is represented by the following formula:

[0013] X is a connecting unit;

[0014] HG is a hydrophilic unit;

[0015] PG is a protecting group;

[0016] P is an enzyme cleavable unit;

[0017] Y is a self-cleavage unit;

[0018] wherein a end is connected to the linker unit Z, and b end is connected to the drug unit D,

[0019] m represents the molar ratio of the drug unit D to Ab (also referred to as DAR, i.e., drug antibody conjugation ratio), which is an integer or a decimal number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) from 1 to 12. When m is a decimal number, it represents the average molar ratio of the drug unit D to Ab.

[0020] In some embodiments, the linker unit Z is selected from or wherein a1 end is connected to Ab, and b1 end is connected to W;

[0021] U is a spacer unit selected from -U1-U2-U3-C(O)-, wherein U1 is selected from -C1-C8alkyl-, -C1-C8alkyl-C3-C8cycloalkyl-, -C6-C10aryl-, -C6-C10aryl-C1-C8alkyl-, -5-6 membered heteroaryl-, -5-6 membered heteroaryl-C1-C8alkyl-, linear or branched heteroalkyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms, linear or branched heteroalkyl-3-8 membered heterocyclyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms; U2 is selected from a bond, -C1-C8alkyl-O-, -O-C1-C8alkyl-, -C1-C8alkyl-C3-C8cycloalkyl-, -C6-C10aryl-, -C6-C10aryl-C1-C8alkyl-, -5-6 membered heteroaryl-, -5-6 membered heteroaryl-C1-C8alkyl-, -C1-C8alkyl-NR 12 aryl-, -C6-C 12 aryl-C1-C8alkyl-, -5-6 membered heteroaryl-, -5-6 membered heteroaryl-C1-C8alkyl-, linear or branched heteroalkyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms, linear or branched heteroalkyl-3-8 membered heterocyclyl of 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms; U2 is selected from a bond, -C1-C8alkyl-O-, -O-C 1-3 alkyl-, -O-, -C(O)-, -NR 1-3 alkyl-, -O-, -C(O)-, -NR 1 -, -NR 1 -C(O)-, -C 1-3 alkyl-NR 1 - or C 1-3 alkyl-NR 1 -C(O)-, R 1 is selected from hydrogen, C1-C6alkyl, C3-C8cycloalkyl, 3-8 membered heterocyclyl, 2-8 membered heteroalkyl; U3 is selected from a bond, C1-C8alkyl, linear or branched heteroalkyl of 2-10 (preferably 2-6) carbon atoms; each of said alkyl, cycloalkyl, aryl, heteroalkyl, heteroaryl, heterocyclyl is optionally substituted with one or more substituents independently selected from C1-C6alkyl, C1-C6alkoxy, hydroxyl, amino, carboxyl, or C3-C8cycloalkyl, said heteroalkyl, heteroaryl, heterocyclyl contains 1-12 (preferably 1-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more of N, O, or S; said “multiple” in the foregoing definition is 2-12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, unless otherwise defined.

[0022] In some embodiments, linker unit Z is selected from or wherein a1 end represents attachment to Ab (in particular, to a thiol of an antibody), b1 end represents attachment to W; wherein, U1 is selected from -C1-C6alkyl-, -C6-C 12 aryl-, -C1-C3alkyl-C3-C6cycloalkyl-, straight chain or branched chain heteroalkyl of 1 to 20 carbon atoms, said heteroalkyl containing from 1 to 12 (preferably 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more of N, O, or S; U2 is selected from a bond, -C 1-3 alkyl-O-, -O-C 1-3 alkyl-, -O-, -C(O)-, -NR 1 -, -NR 1 -C(O)-, -C 1-3 alkyl-NR 1 - or C 1-3 alkyl-NR 1 -C(O)-, R 1 is selected from hydrogen, C1-C3alkyl, C3-C6cycloalkyl, 3-6 membered heterocyclyl, 2-6 membered heteroalkyl; U3 is selected from a bond, C1-C8alkyl, C 1- 3alkyl-O-C 1-3 alkyl, each of said alkyl, heteroalkyl optionally substituted with one or more substituents independently selected from C1-C6alkyl, C1-C6alkoxy, amino, or carboxyl, said heteroalkyl containing from 1 to 12 (preferably 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more of N, O, or S.

[0023] In some embodiments, U1 is selected from -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, phenylene, U2 is selected from a bond, -CH2NHC(O)-, -NHC(O)-, -O-, -CH2O-, -OCH2-, U3 is selected from a bond, -CH2OCH2-, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-.

[0024] In some embodiments, U1 is selected from -(CH2)5-, phenylene, U2 is selected from a bond, -CH2NHC(O)-, -NHC(O)-, -O-, -CH2O-, -OCH2-, U3 is selected from a bond, -CH2OCH2-, -CH2-.

[0025] In some embodiments, U1 is selected from -(CH2)5-, -U2-U3- is selected from the group consisting of: a bond,

[0026] In some embodiments, U1 is selected from the group consisting of: -U2-U3- is selected from the group consisting of: a bond, *the end connected to the phenyl ring.

[0027] In some embodiments, linker unit Z is selected from the group consisting of the following structures:

[0028] wherein a1 end represents connection to a thiol of an antibody, b1 end represents connection to W.

[0029] In some embodiments, linker unit X is selected from the group consisting of: or X1 is selected from the group consisting of -C(O)- or -NR 3 -, k and j are each independently an integer from 0 to 10 (preferably 0 to 6, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), wherein a3 end represents connection to Z, b3 end represents connection to self-immolative unit Y, c1 end represents connection to hydrophilic unit HG; R 2 and R 3 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, 2-8 membered heteroalkyl, each of said alkyl, heteroalkyl optionally substituted with one or more substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, amino, or carboxyl, said heterocyclyl, heteroalkyl containing from 1 to 8 (such as 1, 2, 3, 4, 5, 6, 7, 8) heteroatoms selected from one or more of N, O, or S; said "one or more" in the foregoing definition is 2-8 (such as 2, 3, 4, 5, 6, 7, 8), unless otherwise defined.

[0030] In some embodiments, linker unit X is preferably of the following structure:

[0031] In some embodiments, linker unit X is preferably of the following structure:

[0032] In some embodiments, self-immolative unit Y is selected from the group consisting of the following structures:

[0033] wherein a4 end represents connection to enzyme cleavable unit P, b4 end represents connection to linker unit X, c2 end represents connection to drug unit D.

[0034] In some embodiments, the enzyme-cleavable unit P comprises an enzyme-cleavable polypeptide linked to the self-cleaving unit Y via an amide bond, wherein the polypeptide comprises a dipeptide having the sequence -P2-P1-, a tripeptide -P3-P2-P1-, a tetrapeptide -P4-P3-P2-P1-, a pentapeptide -P5-P4-P3-P2-P1-, and a hexapeptide -P6-P5-P4-P3-P2-P1-, wherein each of P1, P2, P3, P4, P5, and P6 is a natural amino acid or a non-natural amino acid.

[0035] In some embodiments, each of P1, P2, P3, P4, P5 is independently selected from the group consisting of alanine (A), asparagine (N), aspartic acid (D), glutamic acid (E), glycine (G), isoleucine (I), leucine (L), lysine (K), phenylalanine (F), proline (P), threonine (T), valine (V), and citrulline (C).

[0036] In some embodiments, each of P1, P2, P3, P4, P5 is independently selected from the group consisting of alanine (A), asparagine (N), glutamic acid (E), glycine (G), leucine (L), phenylalanine (F), valine (V), and citrulline (C).

[0037] In some embodiments, the enzyme-cleavable unit P is selected from the following structures:

[0038] wherein the a5 end is linked to the hydrophilic unit HG or to a protecting group PG, and the b5 end is linked to the self-cleaving unit Y.

[0039] In some embodiments, PG is selected from the group consisting of an amino protecting group, such as formyl, acetyl, trifluoroacetyl, benzoyl, alkoxycarbonyl (such as methoxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, t-butoxycarbonyl), benzyloxycarbonyl, fluorenylmethoxycarbonyl, and the like.

[0040] In some embodiments, the enzyme-cleavable unit P-PG is selected from the following structures:

[0041] In some embodiments, the enzyme-cleavable unit P-PG is:

[0042] In some embodiments, the hydrophilic unit HG comprises a polyethylene glycol, a peptide chain of natural or non-natural amino acids (such as a polysarcosine), a sugar (such as a monosaccharide, an oligosaccharide, a polysaccharide), and the like.

[0043] In some embodiments, the hydrophilic unit HG is selected from the following structures:

[0044] wherein c3 is attached to a linking unit X or an enzyme cleavable unit P, S1 is selected from a bond, C1-C6 alkylene, 4-12 membered heteroalkylene, R 4 selected from hydrogen, C1-C6 alkyl, 2-6 membered heteroalkyl, HG1 is selected from a linear or branched heteroalkyl comprising 1-50 (preferably 6-24, more preferably 8-24) -OCH2CH2- structural units, or a linear or branched heteroalkyl comprising a 1-50 (preferably 6-24, more preferably 8-24) natural or unnatural amino acid peptide chain (such as a peptide chain comprising 6-50 (preferably 6-24, more preferably 8-24) -N(CH3)CH2C(O)- structural units), or a linear or branched heteroalkyl comprising a monosaccharide, oligosaccharide, or polysaccharide, each of said alkylene, heteroalkylene, alkyl, heteroalkyl being optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, amino, or carboxyl, said heteroalkylene, heteroalkyl containing 1-8 (such as 1, 2, 3, 4, 5, 6, 7, 8) heteroatoms selected from one or more of N, O, or S; said "plurality" in the foregoing definitions is 2-12 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) unless otherwise defined. In some embodiments, the hydrophilic unit HG is selected from wherein HG1 is selected from a linear or branched heteroalkyl comprising 8-24 -OCH2CH2- structural units, or a linear or branched heteroalkyl comprising a 8-12 natural or unnatural amino acid peptide chain (such as a peptide chain comprising 8-12 -N(CH3)CH2C(O)- structural units), or a linear or branched heteroalkyl comprising a monosaccharide, oligosaccharide, or polysaccharide.

[0045] In some embodiments, HG1 is selected from -(CH2CH2O)n-CH2CH2O-, n CH3, -(N(CH3)CH2C(O)) v OH, -CH2C(O)(N(CH3)CH2C(O)) v OH, wherein n and v are each independently selected from an integer of 1-50, preferably an integer of 1-30, further preferably an integer of 6-24, most preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30.

[0046] In some embodiments, the hydrophilic unit HG is selected from the following structures:

[0047] In some embodiments, the hydrophilic unit HG is selected from the following structures:

[0048] In some embodiments, -Z-W- is selected from the following structures:

[0049] In some embodiments, -Z-W- is selected from the following structures:

[0050] In some embodiments, -Z-W- is selected from the following structures:

[0051] In some embodiments, -Z-W- is selected from the following structures:

[0052] In some embodiments, Drug Unit D is selected from an aminohydrogen-containing biologically active drug molecule or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or isotopically-labeled form thereof.

[0053] In some embodiments, Drug Unit D is selected from a camptothecin derivative, eribulin, an auristatin.

[0054] In some embodiments, Drug Unit D is selected from a compound of the following formula:

[0055] wherein c4 is connected to Self-Cleaving Unit Y.

[0056] In some embodiments, biologically active molecule D is selected from:

[0057] In some embodiments, the antibody drug conjugate of Formula (I) and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled forms thereof are selected from the following structures:

[0058] In some embodiments, the antibody drug conjugate of Formula (I) and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled forms thereof are selected from the following structures:

[0059] In some embodiments, the antibody drug conjugate of Formula (I) and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled forms thereof are selected from the following structures:

[0060] In some embodiments, the antibody drug conjugate of Formula (I) and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled forms thereof are selected from the following structures:

[0061] wherein Ab is selected from an antibody or an antigen-binding fragment; m represents the molar ratio of the drug unit D to Ab (also referred to as DAR, i.e., drug antibody conjugation ratio), which is an integer or a decimal number from 1 to 12, for example, an integer or a decimal number from 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; when m is a decimal number, it represents the average molar ratio of the drug unit D to Ab.

[0062] In some embodiments, the antibody moiety Ab of the antibody drug conjugate and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled forms thereof is selected from a monoclonal antibody, a diabody, or an antigen-binding fragment thereof targeting HER2 (ErbB2), GPC3, or CDCP1.

[0063] In some embodiments, Ab is an antibody or an antigen-binding fragment thereof targeting GPC3, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), the heavy chain variable region comprising 3 complementarity determining regions (CDRs), wherein the heavy chain complementarity determining region 1 (HCDR1) amino acid sequence is set forth in SEQ ID NO: 1, the heavy chain complementarity determining region 2 (HCDR2) amino acid sequence is set forth in SEQ ID NO: 2, the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence is set forth in SEQ ID NO: 3; the light chain variable region comprising 3 complementarity determining regions (LCDRs), wherein the light chain complementarity determining region 1 (LCDR1) amino acid sequence is set forth in SEQ ID NO: 4, the light chain complementarity determining region 2 (LCDR2) amino acid sequence is set forth in SEQ ID NO: 5, the light chain complementarity determining region 3 (LCDR3) amino acid sequence is set forth in SEQ ID NO: 6, wherein the CDRs are determined according to the Kabat numbering convention; or

[0064] Ab is an antibody or antigen-binding fragment thereof targeting HER2, comprising a heavy chain variable region comprising 3 complementarity determining regions (CDRs), wherein the heavy chain complementarity determining region 1 (HCDR1) amino acid sequence is set forth in SEQ ID NO: 11, the heavy chain complementarity determining region 2 (HCDR2) amino acid sequence is set forth in SEQ ID NO: 12, and the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence is set forth in SEQ ID NO: 13; and / or a light chain variable region comprising 3 complementarity determining regions (CDRs), wherein the light chain complementarity determining region 1 (LCDR1) amino acid sequence is set forth in SEQ ID NO: 14, the light chain complementarity determining region 2 (LCDR2) amino acid sequence is set forth in SEQ ID NO: 15, and the light chain complementarity determining region 3 (LCDR3) amino acid sequence is set forth in SEQ ID NO: 16, wherein the CDRs are determined according to the Kabat numbering convention; or

[0065] Ab is an antibody or antigen-binding fragment thereof targeting CDCP1, comprising a heavy chain variable region comprising 3 complementarity determining regions (CDRs), wherein the heavy chain complementarity determining region 1 (HCDR1) amino acid sequence is set forth in SEQ ID NO: 21, the heavy chain complementarity determining region 2 (HCDR2) amino acid sequence is set forth in SEQ ID NO: 22, and the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence is set forth in SEQ ID NO: 23; and / or a light chain variable region comprising 3 complementarity determining regions (CDRs), wherein the light chain complementarity determining region 1 (LCDR1) amino acid sequence is set forth in SEQ ID NO: 24, the light chain complementarity determining region 2 (LCDR2) amino acid sequence is set forth in SEQ ID NO: 25, and the light chain complementarity determining region 3 (LCDR3) amino acid sequence is set forth in SEQ ID NO: 26, wherein the CDRs are determined according to the Kabat numbering convention.

[0066] In some embodiments, Ab is an antibody or antigen-binding fragment targeting GPC3, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or

[0067] Ab is an antibody or antigen-binding fragment thereof targeting HER2, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18; or

[0068] Ab is an antibody or antigen-binding fragment thereof targeting HER2, the antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 19, the amino acid sequence of the light chain being as set forth in SEQ ID NO: 20; or

[0069] In some embodiments, Ab is an antibody targeting GPC3, the antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 9, the amino acid sequence of the light chain being as set forth in SEQ ID NO: 10; or

[0070] Ab is an antibody or antigen-binding fragment thereof targeting HER2, the antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 19, the amino acid sequence of the light chain being as set forth in SEQ ID NO: 20; or

[0071] Ab is an antibody or antigen-binding fragment thereof targeting CDCP1, the antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 29, the amino acid sequence of the light chain being as set forth in SEQ ID NO: 30; or

[0072] In some embodiments, Ab is an antibody targeting GPC3, the antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 9, the amino acid sequence of the light chain being as set forth in SEQ ID NO: 10; or

[0073] Codrituzumab comprises 2 identical heavy chains and light chains, the amino acid sequence of the heavy chain being as set forth in SEQ ID NO: 9, the amino acid sequence of the light chain being as set forth in SEQ ID NO: 10.

[0074] Codrituzumab heavy chain amino acid sequence (SEQ ID NO: 9):

[0075] Codrituzumab light chain amino acid sequence (SEQ ID NO: 10):

[0076] wherein the bolded amino acid sequences are the heavy chain / light chain CDR sequences, and the underlined amino acid sequences are the heavy chain / light chain variable region amino acid sequences.

[0077] Codrituzumab heavy chain variable region amino acid sequence (SEQ ID NO: 7):

[0078] Codrituzumab light chain variable region amino acid sequence (SEQ ID NO: 8):

[0079] wherein the heavy chain CDR sequences HCDR1, HCDR2, HCDR3 amino acid sequences are designated as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, respectively, and the light chain CDR sequences LCDR1, LCDR2, LCDR3 amino acid sequences are designated as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively

[0080] In some embodiments, the Ab is an antibody targeting HER2, Trastuzumab.

[0081] Trastuzumab comprises two identical heavy chains and light chains, the heavy chain amino acid sequence is shown as SEQ ID NO: 19, and the light chain amino acid sequence is shown as SEQ ID NO: 20.

[0082] > Trastuzumab heavy chain (SEQ ID NO: 19)

[0083] > Trastuzumab light chain (SEQ ID NO: 20)

[0084] wherein the bolded amino acid sequences are heavy chain / light chain CDR sequences, and the underlined amino acid sequences are heavy chain / light chain variable region amino acid sequences.

[0085] wherein the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 17, and the light chain variable region amino acid sequence is shown as SEQ ID NO: 18.

[0086] > Trastuzumab heavy chain variable region (VH) amino acid sequence (SEQ ID NO: 17)

[0087] > Trastuzumab light chain variable region (VL) amino acid sequence (SEQ ID NO: 18)

[0088] wherein the heavy chain CDR sequences HCDR1, HCDR2, HCDR3 amino acid sequences are designated as SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, respectively, and the light chain CDR sequences LCDR1, LCDR2, LCDR3 amino acid sequences are designated as SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively

[0089] In some embodiments, the Ab is an antibody targeting CDCP1, SYM026.

[0090] SYM026 comprises 2 identical heavy chains and light chains, the heavy chain amino acid sequence is shown as SEQ ID NO: 29, and the light chain amino acid sequence is shown as SEQ ID NO: 30.

[0091] > SYM026 heavy chain (SEQ ID NO: 29)

[0092] > SYM026 light chain (SEQ ID NO: 30)

[0093] wherein the bolded amino acid sequences are heavy chain / light chain CDR sequences, and the underlined amino acid sequences are heavy chain / light chain variable region amino acid sequences.

[0094] wherein the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 27, and the light chain variable region amino acid sequence is shown as SEQ ID NO: 28.

[0095] > SYM026 heavy chain variable region (VH) amino acid sequence (SEQ ID NO: 27)

[0096] > SYM026 light chain variable region (VL) amino acid sequence (SEQ ID NO: 28)

[0097] wherein the heavy chain CDR HCDR1, HCDR2, HCDR3 amino acid sequences are SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, respectively, and the light chain CDR LCDR1, LCDR2, LCDR3 amino acid sequences are SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

[0098] In some embodiments, the antibody moiety Ab of the antibody drug conjugate and its tautomers, meso forms, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotopically-labeled forms can be modified, for example, one or more amino acid changes, additions or deletions.

[0099] In another aspect, the present application provides a linker-drug compound represented by formula (II) or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotopically-labeled form thereof:

[0100] wherein Z' is a precursor of a linker unit capable of being converted to Z of formula (I) to form a covalent bond to Ab of formula (I);

[0101] W, D and Ab are as defined in formula (I).

[0102] In some embodiments, the precursor of a linker unit Z' is selected from or wherein b1 is attached to W, and U is as defined in formula (I).

[0103] In some embodiments, the linker unit Z is selected from wherein b1 is attached to W, and U1, U2, U3 are as defined in formula (I).

[0104] In some embodiments, the precursor of a linker unit Z' is selected from the following structures:

[0105] In some embodiments, the linker-drug compounds of formula II and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled versions thereof are selected from the following structures:

[0106] In some embodiments, the linker-drug compounds of formula II and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled versions thereof are selected from the following structures:

[0107] In some embodiments, the linker-drug compounds of formula II and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled versions thereof are selected from the following structures:

[0108] In some embodiments, the linker-drug compounds of formula (II) and tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled versions thereof are selected from the following structures:

[0109] In some embodiments, the present application provides linkers of formula (III) and tautomers, stereoisomers, solvates, or isotopically-labeled versions thereof, selected from the following structures:

[0110] wherein Z' is a first linker unit precursor, which is capable of being converted to Z of formula (I), thereby forming a covalent bond to Ab of formula (I);

[0111] W' is a second linker unit precursor, which is capable of being converted to W of formula (I), thereby forming a covalent bond to D of formula (I);

[0112] The definitions of the foregoing Z', Z, W, D and Ab are the same as those of formula (I) or formula (II).

[0113] In some embodiments, W' of formula (III) is represented by the following formula:

[0114] Y' is selected from the following structures:

[0115] The definitions of the foregoing a, X, HG, PG, P, a4, b4 are the same as those of formula (I).

[0116] In some embodiments, the linker represented by formula (III) and its tautomers, stereoisomers, solvates or isotopically-labeled versions thereof is selected from the following structures:

[0117] In some embodiments, the linker represented by formula (III) and its tautomers, stereoisomers, solvates or isotopically-labeled versions thereof is selected from the following structures:

[0118] Another aspect of the present application provides a pharmaceutical composition comprising the antibody drug conjugate of formula (I) and its tautomers, stereoisomers, prodrugs thereof, pharmaceutically acceptable salts thereof, solvates thereof or isotopically-labeled versions thereof, or the linker-drug having the structure of formula (II) and its tautomers, stereoisomers, prodrugs thereof, pharmaceutically acceptable salts thereof, solvates thereof or isotopically-labeled versions thereof, and optionally one or more pharmaceutical excipients.

[0119] Another aspect of the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody drug conjugate of formula (I) and its tautomers, stereoisomers, prodrugs thereof, pharmaceutically acceptable salts thereof, solvates thereof or isotopically-labeled versions thereof, or the linker-drug having the structure of formula (II) and its tautomers, stereoisomers, prodrugs thereof, pharmaceutically acceptable salts thereof, solvates thereof or isotopically-labeled versions thereof, and optionally one or more pharmaceutical excipients.

[0120] In some embodiments, the present application provides the use of an antibody drug conjugate represented by Formula (I) and its tautomer, mesomer, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotopically-labeled for treating cancer. Or the use of a linker-drug represented by Formula (II) and its tautomer, stereoisomer, prodrug thereof, pharmaceutically acceptable salt, solvate or isotopically-labeled thereof for treating cancer.

[0121] In some embodiments, the present application provides the use of an antibody drug conjugate represented by Formula (I) and its tautomer, mesomer, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotopically-labeled for treating cancer. Or the use of a linker-drug represented by Formula (II) and its tautomer, stereoisomer, prodrug thereof, pharmaceutically acceptable salt, solvate or isotopically-labeled thereof for treating cancer.

[0122] In some embodiments, the cancer includes liver cancer, ovarian cancer, colorectal cancer, breast cancer, etc.

[0123] Abbreviations and definitions

[0124] The following terms used in the present application have the following meanings, unless otherwise indicated.

[0125] When a trademark name is used in the present application, unless otherwise indicated in the context, the trademark name includes the product formula, generic drug and active pharmaceutical ingredient of the product of the trademark name.

[0126] The linker-drug compound described in the present application is connected to the antibody by a coupling method conventional in the art, including: lysine coupling, inter-heavy chain reducing disulfide bond coupling and directed coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6: 15-17; McCombs J R, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 2015, 17: 339-351). The present application is preferably connected by inter-heavy chain reducing disulfide bond coupling, i.e. the reaction of the thiol group (sulfur atom of cysteine residue) formed after one or more of the inter-heavy chain disulfide bond sites (two sites between heavy chains, two sites between light and heavy chains) is reduced.

[0127] The term "tautomer" refers to structural isomers that can interconvert by a low energy barrier. If tautomerism is possible (as in solution), a chemical equilibrium of tautomers can be achieved. For example, prototropic tautomers (also known as proton-shift tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by reorganization of some bonding electrons.

[0128] The term "stereoisomer" refers to compounds which have the same chemical constitution, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans isomers), atropisomers, and the like. Any mixture of stereoisomers can be separated into its individual isomers based on the differences in their physical and chemical properties under given conditions, for example, by chromatography and / or fractional crystallization.

[0129] The term "prodrug" refers to a drug which is converted into the parent drug in vivo. One example, without limitation, of a prodrug is any compound of the application which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane and release of the active drug only in the cytoplasm of the cell. Another example of a prodrug can be a short peptide (polyaminoacid) bound to an acid group, wherein the peptide is metabolized to reveal the active moiety.

[0130] The term "solvate" means a physical association of a compound of this application with one or more solvent molecules (either organic or inorganic). This physical association can include hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of the soluble compound. The solvent molecule can be present in a constant or variable number in the solvate. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods for solvation are generally known in the art.

[0131] The term "isotopic derivative" means that the compounds of the application can exist in isotopically-labelled or enriched forms, containing one or more atoms which have an atomic mass or mass number different from the atomic mass or mass number of the most abundant prime isotope of that atom commonly found in nature. The isotopes can be radioactive or non-radioactive. The isotopes typically used as labels are: hydrogen isotopes, 2 H and 3 H; carbon isotopes: 13 C and 14 C; chlorine isotopes: 35 Cl and 37 Cl; fluorine isotopes:18 F; iodine isotopes: 123 I and 125 I; nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotopically-labeled compounds can be used to study the distribution of the pharmaceutical molecule in tissue.

[0132] The term "plurality" in the term "one or more" includes two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0133] The term "heteroatom" means a nitrogen, oxygen, sulfur, or halogen atom.

[0134] The term "C m-n " or "C m -C n " have the same meaning, i.e., the moiety has an integer number of carbon atoms in the range of m to n. For example, "C 1-3 " means that the group can have 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0135] The term "- (CH2) n -" means that the moiety has n -CH2- attached groups, e.g., when the number of linking groups is 0, such as in "-(CH)0-", it means that the linking group is a covalent bond.

[0136] The term "alkyl" means a monovalent saturated aliphatic hydrocarbon group including straight-chain or branched-chain groups, preferably containing 1-20 carbon atoms (i.e., C 1-10 alkyl), further preferably containing 1-8 carbon atoms (i.e., C 1-8 alkyl), more preferably containing 1-6 carbon atoms (i.e., C 1-6 alkyl) or 1-3 carbon atoms (i.e., C 1-3 alkyl). For example, "C 1-6 alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5, or 6), examples including but not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and the like.

[0137] The term "alkoxy" refers to -O-alkyl, the alkyl groups being defined as above, i.e. containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, even more preferably 1 to 6 carbon atoms (in particular 1, 2, 3, 4, 5 or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propyloxy, isopropoxy, butoxy, 1 -methylpropyloxy, 2-methylpropyloxy, t-butoxy, pentyloxy, 1 -methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1 -dimethylpropyloxy, 1,2-dimethylpropyloxy, 2,2-dimethylpropyloxy, 1 -ethylpropyloxy and the like.

[0138] The term "halogen" refers to F, CI, Br, I.

[0139] The term "aryl" denotes monocyclic, bicyclic and tricyclic aromatic carbocyclic ring systems containing 6 to 16 carbon atoms, or 6 to 14 carbon atoms, or 6 to 12 carbon atoms, or 6 to 10 carbon atoms, preferably 6 to 10 carbon atoms, such as C6-C10-aryl. Examples of aryl groups can include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl or pyrenyl and the like. 12 The term "aryl" denotes monocyclic, bicyclic and tricyclic aromatic carbocyclic ring systems containing 6 to 16 carbon atoms, or 6 to 14 carbon atoms, or 6 to 12 carbon atoms, or 6 to 10 carbon atoms, preferably 6 to 10 carbon atoms, such as C6-C10-aryl. Examples of aryl groups can include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl or pyrenyl and the like.

[0140] The term "heteroaryl" denotes aromatic monocyclic or polycyclic ring systems containing a 5- to 14-membered structure, or preferably a 5- to 10-membered structure, or preferably a 5- to 8-membered structure, or more preferably a 5- to 6-membered structure, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms being independently selected from O, N or S, the number of heteroatoms preferably being 1, 2 or 3. Examples of heteroaryl groups include, but are not limited to, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiadiazolyl, triazinyl, phtalazinyl, quinolinyl, isoquinolinyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidinyl, benzopyrazinyl, benzimidazolyl, benzophtalazinyl, pyrrolo[2,3-b]pyridyl, imidazo[1,2-a]pyridyl, pyrazolo[1,5-a]pyridyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridyl and the like.

[0141] The term "cycloalkyl" refers to a carbocyclic ring which is fully saturated and can exist as a monocyclic, bridged or spirocyclic ring. Preferably, it contains 3 to 12 carbon atoms (i.e. C3-Ci2-cycloalkyl), more preferably 3 to 10 carbon atoms (C3-Ci0-cycloalkyl), even more preferably 3 to 8 carbon atoms (C3-C8-cycloalkyl), and most preferably 3 to 6 carbon atoms (C3-C6-cycloalkyl). 12 The term "cycloalkyl" refers to a carbocyclic ring which is fully saturated and can exist as a monocyclic, bridged or spirocyclic ring. Preferably, it contains 3 to 12 carbon atoms (i.e. C3-Ci2-cycloalkyl), more preferably 3 to 10 carbon atoms (C3-Ci0-cycloalkyl), even more preferably 3 to 8 carbon atoms (C3-C8-cycloalkyl), and most preferably 3 to 6 carbon atoms (C3-C6-cycloalkyl). 10Cycloalkyl groups, preferably with 3-8 carbon atoms (C3-C8 cycloalkyl), 3-7 carbon atoms (C3-C7 cycloalkyl), 4-6 carbon atoms (C4-C6 cycloalkyl), or 5-6 carbon atoms (C5-C6 cycloalkyl). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, etc.

[0142] The term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight-chain or branched alkylene having 1 to 8 carbon atoms.

[0143] The terms "-alkyl-", "-C" m-n "alkyl-" refers to alkylene.

[0144] In various parts of this invention, various linking substituents (e.g., W, U1, U2, U3, etc.) are described. Those skilled in the art will understand that when a linking group is clearly required in the compound structure, the Markush variables listed for that group should be understood as linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl," it should be understood that "alkyl" or "aryl" represents a linked alkylene group or an arylene group, respectively. Therefore, when used as a linking group, "*-" is equivalent to "*-" or "-alkyl-", for example, "C1-C8 alkylene" is equivalent to "C1-C8 alkyl".

[0145] The term "heterocyclic group" refers to a non-aromatic ring that is fully saturated or partially unsaturated (not fully unsaturated heteroaromatics) and can exist as a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3- to 7-membered ring containing 1 to 3 heteroatoms independently selected from sulfur, oxygen, and / or nitrogen (preferably 1 or 2 heteroatoms, but excluding -OO-, -OS-, or -SS- portions). Non-limiting examples of heterocyclic groups include, but are not limited to, ethylene oxide, tetrahydrofuranyl, dihydrofuranyl, pyrrolyl, N-methylpyrrolyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothiophene, etc.

[0146] The term "heteroalkyl" by itself or in combination with another term means a stable straight-chain, branched-chain, or combination alkyl radical, or the like, consisting of a number of carbon atoms and at least one heteroatom. The number of carbon atoms can be 1 to 50 (preferably 1 to 20, more preferably 1 to 12, most preferably 1 to 8), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. The heteroalkyl group can optionally contain one, two, or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) heteroatoms selected from N, O, S (or interpreted as optional insertion of heteroatoms into optionally C-C and C-H bonds of the alkyl group). The heteroatoms O, N, and S can be located at any interior position of the heteroalkyl group or at the position of the attachment of the alkyl group to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3. Up to two heteroatoms can be consecutive, such as -CH2-NH-OCH3. In another example, the heteroalkyl group is a 2-50 membered heteroalkyl (or 2-20 membered, 2-12 membered, 4-12 membered, 2-10 membered, 2-8, 2-6 membered heteroalkyl), where 2-50 members represent the number of carbon and heteroatoms excluding hydrogen, the number of carbon atoms and the number of heteroatoms being as recited above. The term "heteroalkyl" includes "heteroalkylene" where appropriate, such as where the heteroalkyl group is divalent or where the substituent is attached to a ring.

[0147] The term "derivative" means a compound formed when an atom or group of atoms in a parent compound is replaced by another atom or group of atoms, which is referred to as a derivative of the parent compound.

[0148] The phrase "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker-drug, or an antibody-linker-drug conjugate). The compound can contain at least one amino, imino, hydroxy, or carboxyl group and thus can form a pharmaceutically acceptable addition salt with a corresponding acid or base. Exemplary salts include, but are not limited to: sulfate, trifluoroacetate, citrate, acetate, oxalate, hydrochloride, hydrobromide, hydroiodide, nitrate, bisulfate, phosphate, acid phosphate (-H2PO4), phosphite, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, ammonium salt, calcium salt, and the like. In addition, pharmaceutically acceptable salts have more than one charged atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter examples. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter atoms.

[0149] The term "amino acid" refers to a naturally occurring amino acid or a non-naturally occurring amino acid represented by NH2-C(R'R")-C(=0)OH, wherein each of R' and R" is independently hydrogen, optionally substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, or heteroalkyl, or R" and the N-terminal nitrogen atom can be taken together to form a heterocyclic ring, e.g., proline.

[0150] The term "peptide" refers to a short chain of amino acid monomers linked by peptide (amide) bonds.

[0151] The term "tumor" refers to a new growth of tissue that results from abnormal cell growth, which can be a malignant or non-malignant process. Tumors exhibit relatively rapid and uncontrolled growth, and can be either benign or malignant.

[0152] The term "linker" refers to a chemical moiety fragment that links one end to an antibody and the other end to a drug unit D, represented by -Z-W-.

[0153] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site within a variable region of the immunoglobulin molecule, represented by Ab. Encompassed are intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies (such as bispecific antibodies, biparatopic antibodies, etc.), multivalent antibodies (such as triavalent, tetravalent, etc. antibodies having three, four or more antigen binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins incorporating an antigen recognition site, and any other modified immunoglobulin molecule that exhibits the desired biological activity.

[0154] The term "heavy chain variable region (VH)" refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.

[0155] The term "light chain variable region (VL)" refers to the amino-terminal variable region domain of an immunoglobulin light chain.

[0156] The term "CDR (complementarity determining region)" refers to the amino acid sequences that together define the antigen binding site of an antibody. Because of the structural complementarity between the antibody combining site and the antigenic determinant, the specificity of an antibody is determined by the chemical composition and spatial geometry of the antibody binding site. The antibody combining site is composed of a number of contributions from the side chains of the residues in the so-called hypervariable or complementarity determining regions (CDRs), interspersed among less variable regions called framework regions (FRs). The light (L) and heavy (H) chains each have three CDRs, designated CDR1, CDR2, and CDR3, in the light chain and CDR1, CDR2, and CDR3, in the heavy chain. Thus, the antigen binding site of a conventional antibody includes six CDRs, comprising the CDR sets from each of the heavy and light chain variable regions. Each VH and VL is composed of three CDRs and four FRs, in the following arrangement from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0157] The term "antigen binding fragment" refers to antibody fragments such as Fv, Fab, F(ab')2 or Fab', or any fragment that should be able to increase the half-life by chemical modification or by incorporation into liposomes, such as the addition of poly(alkylene)glycols such as polyethylene glycol ("PEGylation") (PEGylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" is polyethylene glycol), which have CD73 binding activity. Preferably, the functional fragment will be composed of or comprise partial sequences of the heavy or light chain variable region of the antibody from which it is derived, which are sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, such a functional fragment will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the sequence of the antibody from which it is derived. Examples of antigen binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments with a VL-CL chain and a VH-CH1 chain; (2) F(ab')2 fragments, which can be bivalent fragments with two Fab' fragments connected by a disulfide bridge in the hinge region (i.e. a dimer of Fab'); (3) Fv fragments with the VL and VH domains of a single arm of an antibody.

[0158] As used herein, the term "bispecific antibody" refers to whole or intact antibody molecules (e.g., IgM, IgG (including IgGl, IgG2, IgG3, and IgG4), IgA, IgD, or IgE) and any fragments thereof that bind to two or more different proteins, at least two of which are C5a, C5b, or C5aR (see above). The term bispecific antibody includes, for example, chimerized or chimeric antibodies, humanized antibodies, de-immunized human antibodies, and fully human antibodies. Bispecific antibodies also include, for example, F(ab')2 fragments or conjugates of two or more monospecific antibody fragments (e.g., two or more scFv, Fab, Fab', or Fd immunoglobulin fragments). In addition, bispecific intrabodies, minibodies, triabodies, and diabodies (see, e.g., Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1): 177-189; Poljak (1994) Structure 2(12): 1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283, the disclosures of each of which are incorporated herein by reference in their entireties) are also included in the definition of bispecific antibodies and are compatible for use in the methods described herein. Also encompassed by the term bispecific antibody are tandem single-chain antibodies, single-chain diabodies, tandem single-chain diabodies, and fusion proteins containing a single-chain diabody and at least a portion of an immunoglobulin heavy chain constant region (e.g., a CHI or CH3 region of a heavy chain polypeptide), as described in, e.g., Kontermann (2005) Acta Pharmacologica Sinica 26(1): 1-9; Kufer et al. (2004) Trends Biotechnol 22:238-244; and Kriangkum et al. (2001) Biomol Eng 18:31-40.

[0159] The term "antibody drug conjugate" refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug through a stable linking unit. BRIEF DESCRIPTION OF DRAWINGS

[0160] Figure 1 is a graph showing the effect of antibody drug conjugate on body weight change (left panel) and tumor growth inhibition (right panel) in HepG2 xenograft model animals

[0161] Figure 2 is a graph showing the effect of antibody drug conjugate on body weight change (left panel) and tumor growth inhibition (right panel) in Huh-7 xenograft model animals

[0162] Figure 3 is the effect of antibody drug conjugate on the body weight change (left panel) and tumor growth inhibition (right panel) of Hep3B xenograft model animals

[0163] Figure 4 is the effect of antibody drug conjugate on the body weight change (left panel) and tumor growth inhibition (right panel) of SK-OV-3 xenograft model animals DETAILED DESCRIPTION

[0164] The application will be further illustrated by the following examples, which are not intended to limit the scope of the application. The test methods used in the following examples, unless otherwise stated, were carried out under conventional conditions or as recommended by the manufacturer. All percentages, proportions, ratios, or fractions, unless otherwise stated, are by weight.

[0165] Explanation of Abbreviations

[0166] Example 1: Synthesis of Compound 6

[0167] Compound 1 (28.5 g), potassium hydroxide (12.7 g) and ethanol (400 mL) were added to a reaction flask and stirred to dissolve at room temperature. After the addition of carbon disulfide (17 g) slowly, the reaction was refluxed for 5 hours. After the removal of solvent under reduced pressure, water (50 mL) was added, the pH was adjusted to 6 with dilute hydrochloric acid, and extracted with dichloromethane (100 mL x 3), the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 2 (32.8 g). MS (ESI): (M+H) + Calculated 194.0, found 194.1.

[0168] Compound 2 (3.86 g), triethylamine (2.42 g) and tetrahydrofuran (36 mL) were added to a reaction flask, and methyl iodide (3.12 g) was added to the above reaction solution. After stirring at 25°C for 1.5 hours, the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 3 (2.73 g). MS (ESI): (M+H) + Calculated 208.0, found 208.2.

[0169] Compound 3 (2.20 g), diglycolic anhydride 4 (1.35 g), triethylamine (2.14 g) and tetrahydrofuran (30 mL) were added to a reaction flask, and stirred at 25°C for 1.5 hours. The solvent was removed under reduced pressure, and the residue was added to diethyl ether, filtered, and washed with water and diethyl ether to obtain compound 5 (3.4 g). MS (ESI): (M+H) + Calculated 324.1, found 324.2.

[0170] Compound 5 (3.4 g) and glacial acetic acid (20 mL) were added to a reaction flask, after dissolution, potassium permanganate (2.48 g) was added at 0 °C, and the reaction was allowed to proceed at room temperature for 1 hour, saturated sodium sulfite solution was added until the solution turned colorless, and extraction was performed with ethyl acetate (100 mL x 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 6 (2.4 g). MS (ESI): (M+H) + Calculated 356.0, found 356.0.

[0171] Example 2: Synthesis of compound 12

[0172] Compound 7 (28.7 g), potassium hydroxide (12.7 g), and ethanol (400 mL) were added to a reaction flask, and stirring was performed at room temperature until dissolution. After the slow addition of carbon disulfide (17 g), the reaction was allowed to proceed at reflux for 5 hours. After the removal of the solvent under reduced pressure, water (50 mL) was added, the pH was adjusted to 6 with dilute hydrochloric acid, extraction was performed with dichloromethane (100 mL x 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 8 (33.0 g). MS (ESI): (M+H) + Calculated 195.0, found 195.1.

[0173] Compound 8 (19.4 g) was dissolved in anhydrous methanol (200 mL), N,N- diisopropylethylamine (32.3 g) was added dropwise to the reaction solution at room temperature, and the reaction solution was stirred for 15 minutes before the addition of iodomethane (14.2 g), and the reaction solution was replaced with nitrogen three times. The mixture was stirred at room temperature for 45 minutes. After the reaction was completed, the mixture was concentrated under reduced pressure in a 45 °C water bath, diluted with water (300 mL), and then extracted with ethyl acetate (500 mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain white solid compound 9 (17.5 g). MS (ESI): (M+H) + Calculated 209.0, found 209.1.

[0174] Compound 9 (16.6 g) and tert-butyl 2-bromoacetate (15.8 g) were dissolved in anhydrous tetrahydrofuran (100 mL), and triethylamine (16.2 g) was added to the reaction solution at room temperature. The mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was separated and purified by column chromatography to obtain white solid compound 10 (20.1 g) under reduced pressure. MS (ESI): (M+H) + Calculated 323.1, found 323.1.

[0175] Compound 10 (16.1 g) was dissolved in anhydrous dichloromethane (100 mL), trifluoroacetic acid (57.1 g) was added dropwise into the reaction solution at 0 °C, and the reaction solution was replaced with nitrogen three times. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound 11 (12.5 g) in the form of light yellow oil. MS (ESI): (M+H) + Calcd 267.0, Found 267.1.

[0176] Compound 11 (5.32 g,) and glacial acetic acid (40 mL) were added to a reaction bottle, and after dissolution, potassium permanganate (4.74 g) was added at 0 °C, and the reaction was allowed to proceed at room temperature for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless, and extraction was performed with ethyl acetate (100 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 12 (4.17 g). MS (ESI): (M+H) + Calcd 299.0, Found 299.1.

[0177] Example 3: Synthesis of compound 15

[0178] Compound 13 (35.0 g) was dissolved in anhydrous dichloromethane (350 mL), and EDCI (24.7 g) and N-hydroxysuccinimide (13.7 g,) were slowly added to the reaction solution in batches at 0 °C. The mixture was stirred at room temperature for 4.5 hours. After the reaction was completed, the mixture was added to water (250 mL), and then extraction was performed with dichloromethane (200 mL x 3) to obtain the combined organic phase, which was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography to obtain compound 14 (39.1 g) in the form of light yellow oil. MS (ESI): (M+H) + Calcd 451.2, Found 451.3.

[0179] Compound 14 (39.1 g) was dissolved in ethylene glycol dimethyl ether (200 mL) and water (200 mL), and compound L-alanine (11.7 g) was added to the reaction solution at 25 °C. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product, which was separated and purified by column chromatography to obtain compound 15 (21.0 g) in the form of light yellow solid. MS (ESI): (M+H) + Calcd 425.2, Found 425.1.

[0180] Example 4: Synthesis of compound 22

[0181] Compound 16 (10.0 g) was dissolved in anhydrous N,N-dimethylformamide (200 mL) and anhydrous methanol (200 mL), triethylamine (12.0 g), palladium acetate (888 mg) was added to the reaction solution at room temperature, the reaction system was replaced with carbon monoxide for three times. The mixture was stirred at 80 °C under carbon monoxide (50 Psi) atmosphere for 12 hours. TLC showed that the starting material was completely consumed. The mixture was cooled to room temperature, water (200 mL) was added to quench, then filtered, the filtrate was extracted with ethyl acetate (200 mL x 3), the combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography to obtain compound 17 (5.08 g) as a light yellow solid. MS (ESI): (M+H) + Calcd 233.0, Found 233.1.

[0182] Compound 17 (5.08 g) was dissolved in anhydrous methanol (60 mL), platinum dioxide (586 mg) was added to the reaction solution at room temperature, the reaction system was replaced with hydrogen for three times. The mixture was stirred at 30 °C under hydrogen (50 Psi) atmosphere for 12 hours. TLC showed that the starting material was completely consumed. The mixture was cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure to obtain compound 18 (3.94 g) as a light yellow solid. MS (ESI): (M+H) + Calcd 207.1, Found 207.2.

[0183] Compound 18 (2.50 g) was dissolved in anhydrous methanol (50 mL), di-tert-butyl dicarbonate (2.65 g) was added to the reaction solution at 0 °C, the reaction solution was warmed to 25 °C and stirred for 4 hours. After the reaction was completed, the mixture was added to water (100 mL) to quench, extracted with ethyl acetate (50 mL x 3) and the combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography to obtain compound 19 (2.86 g) as a white solid. MS (ESI): (M+H) + Calcd 307.1, Found 307.2.

[0184] Compound 19 (2.86 g) was dissolved in anhydrous tetrahydrofuran (30 mL) and methanol (2 mL), the reaction solution was replaced with nitrogen for three times, lithium borohydride solution (23.4 mL, 46.8 mmol, 2M) was slowly added to the reaction solution at 0 °C. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was cooled to 0 °C, water (30 mL) was slowly added to quench, then extracted with ethyl acetate (50 mL x 3), the combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain the crude product, which was separated and purified by column chromatography to obtain compound 20 (1.58 g) as a light yellow solid. MS (ESI): (M+H)+ Calculated value: 279.2, Experimental value: 279.2.

[0185] Compound 20 (1.50 g) was dissolved in anhydrous dichloromethane (20 mL), and compound 21 (2.52 g) and EEDQ were added at 0 °C.

[0186] (1.33 g) was added sequentially to the reaction solution, and the reaction solution was purged with nitrogen three times. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was quenched in water (20 mL), then extracted with ethyl acetate (10.0 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain a white solid compound 22 (2.04 g). MS (ESI): (M + H) + The calculated value is 686.3, and the experimental value is 686.4.

[0187] Compound 22 (500 mg) was dissolved in tetrahydrofuran (10 mL), and DBU (8.93 mg) was added under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the mixture was slurried with petroleum ether (30 mL), filtered, and dried to give a white solid compound 23 (310 mg). MS (ESI): (M+H) + Calculated value: 464.3, experimental value: 464.4.

[0188] Compound 23 (310 mg) was dissolved in DMF (5 mL). Compound 15 (284 mg) was added to the solution, followed by DMTMM (398 mg) at 0 °C. The mixture was stirred for 10 minutes, then brought to room temperature and stirred overnight. After the reaction was complete, water (15 mL) was added and the mixture was stirred and filtered. The mixture was washed three times with petroleum ether (20 mL × 3). The filter cake was dried under vacuum to obtain compound 24 (403 mg). MS (ESI): (M + H) + Calculated value: 870.4; Experimental value: 870.5.

[0189] Compound 24 (400 mg) was dissolved in tetrahydrofuran (5 mL), and DBU (55.99 mg) was added under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the mixture was slurried with petroleum ether (30 mL), filtered, and dried to give a white solid compound 25 (274 mg). MS (ESI): (M+H) + The calculated value is 648.4, and the experimental value is 648.6.

[0190] Compound 25 (274 mg) and AcOSu (100 mg) were dissolved in tetrahydrofuran (10 mL), DIEA (109 mg) was added at 0 °C, stirred for 10 minutes, then returned to room temperature, stirred for 4 hours, after the reaction was completed, petroleum ether (30 mL) was added to the slurry, filtered, dried to obtain white solid compound 26 (286 mg). (M+H) + Calculated 690.4, found 690.5.

[0191] Compound 26 (265 mg) was dissolved in dichloromethane (5 mL), TBSOTf (203 mg) was added at 0 °C, and the reaction was carried out at 0 °C for 2 hours. After the reaction was completed, it was directly passed through a reversed-phase silica gel column, concentrated, and dried to obtain white solid compound 27 (203 mg). (M+H) + Calculated 590.3, found 590.4.

[0192] Example 5: Synthesis of compound 30

[0193] Compound 28 (30 g) and propargylamine (3.88 g) were dissolved in tetrahydrofuran (300 mL), DIEA (11.8 g) and EDCI (17.5 g) were added at room temperature, and the mixture was stirred at room temperature for 12 hours under nitrogen atmosphere. After the reaction was completed, the reaction solution was added to water (200 mL) for quenching, and extracted with ethyl acetate (200 mL x 3). The combined organic phase was washed with saturated brine (600 mL), dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography, and concentrated under reduced pressure to obtain yellow solid compound 29 (10.4 g). (M+H) + Calculated 463.2, found 463.3.

[0194] Compound 29 (10.4 g) was dissolved in dichloromethane (90.0 mL), trifluoroacetic acid (54.3 g) was slowly added at 0 °C, and the mixture was stirred at 20 °C for 2 hours under nitrogen atmosphere. After the reaction was completed, the reaction solution was slowly poured into ice water (200 mL) and stirred, and a solid was precipitated during the stirring process. The solid was filtered and concentrated under reduced pressure to obtain white solid compound 30 (6.02 g). (M+H) + Calculated 407.2, found 407.3.

[0195] Example 6: Synthesis of compound 35

[0196] Compound 27 (200 mg) was dissolved in DMF (5 mL), compound 30 (137 mg) was added, HATU (155 mg) and NMM (85.7 mg) were added at 0 °C, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, column chromatography was used for separation and purification to obtain compound 31 (252 mg) in the form of a white solid. MS (ESI): (M+H) + Calculated 978.5, found 978.5.

[0197] Compound 31 (250 mg) was dissolved in tetrahydrofuran (5 mL), compound 32 (103 mg) was added, pyridine (40 mg) was added at 0 °C, and the mixture was slowly warmed to 60 °C and stirred for 2 h. After the reaction was completed, column chromatography was used for separation and purification to obtain compound 33 (175 mg) in the form of a white solid. MS (ESI): (M+H) + Calculated 1143.5, found 1143.6.

[0198] Compound 33 (175 mg) was dissolved in DMF (5 mL), irinotecan methanesulfonate (81 mg) was added, DIEA (59 mg) was added at 0 °C, the mixture was stirred at room temperature overnight, and column chromatography was used for separation and purification to obtain compound 34 (191 mg) in the form of a yellow solid. MS (ESI): (M+H) + Calculated 1439.6, found 1439.7.

[0199] Compound 34 (191 mg) was dissolved in tetrahydrofuran (10 mL), DBU (16 mg) was added at 0 °C under a nitrogen atmosphere, the mixture was stirred at room temperature for 0.5 h, and after the reaction was completed, the mixture was slurried with petroleum ether (10 mL), filtered, and dried to obtain compound 35 (146 mg). MS (ESI): (M+H) + Calculated 1217.5, found 1217.6.

[0200] Example 7: Synthesis of compound 37

[0201] Compound 35 (85 mg) was dissolved in DMF (5 mL), EDCI (16 mg), HOBT (9 mg), and compound 6 (25 mg) were added at 0 °C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, column chromatography was used for separation and purification to obtain compound 36 (82 mg) in the form of a yellow solid. MS (ESI): (M+H) + Calculated 1554.6, found 1554.7.

[0202] Compound 36 (82 mg) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added at 0 °C under nitrogen atmosphere, and the reaction was carried out at 0 °C for 4 hours. After the reaction was completed, compound 37 (78 mg) was obtained by column chromatography. MS (ESI): (M+H) + Calculated 1498.5, found 1498.6.

[0203] Example 8: Synthesis of compound LD1

[0204] Compound 37 (40 mg) was dissolved in tetrahydrofuran (2 mL), water (4 mL), and DMF (1 mL), and compound 38 (18.7 mg) was added under nitrogen atmosphere. After blowing nitrogen for 15 minutes, CuBr (0.5 mg) and THPTA (0.7 mg) were added, and the reaction was carried out at 25 °C for 0.5 hours. After the reaction was completed, compound LD1 (12 mg) was obtained by reverse phase preparative chromatography. MS (ESI): (M+H) + Calculated 2083.9, found 2084.0.

[0205] Example 9: Synthesis of compound LD2

[0206] Compound 37 (40 mg) was dissolved in tetrahydrofuran (2 mL), water (4 mL), and DMF (1 mL), and compound 39 (35.6 mg) was added under nitrogen atmosphere. After blowing nitrogen for 15 minutes, CuBr (0.5 mg) and THPTA (0.7 mg) were added, and the reaction was carried out at 25 °C for 0.5 hours. After the reaction was completed, compound LD2 (21 mg) was obtained by reverse phase preparative chromatography. MS (ESI): (M+H) + Calculated 2612.2, found 2612.3.

[0207] Example 10: Synthesis of compound LD3

[0208] Compound 37 (40 mg) was dissolved in tetrahydrofuran (2 mL), water (4 mL), and DMF (1 mL), and compound 40 (35.6 mg) was added under nitrogen atmosphere. After blowing nitrogen for 15 minutes, CuBr (0.5 mg) and THPTA (0.7 mg) were added, and the reaction was carried out at 25 °C for 0.5 hours. After the reaction was completed, compound LD3 (21 mg) was obtained by reverse phase preparative chromatography. MS (ESI): (M+H) + Calculated 2309.9, found 2310.0.

[0209] Example 11: Synthesis of compounds LD4-18

[0210] The synthesis route of compound LD4-18 refers to the synthesis of compound LD1. The structures of Con1-2, HDP1-3, Pay1-3 are shown as follows:

[0211] Example 12: General method for ADC sample preparation and DAR determination

[0212] The antibody with known sequence can be obtained by protein expression method which is well known to those skilled in the art. After fermentation broth, higher purity sample can be obtained by affinity chromatography, ion chromatography and other steps.

[0213] The antibody sample Codrituzumab (targeting GPC3) is diluted to about 10 mg / mL with phosphate buffer solution at pH 7.0, and the antibody concentration is detected by ultraviolet spectrophotometry. 1 mL of the antibody solution after liquid exchange is placed in a 2 mL centrifuge tube, 5 mM TCEP (Adamas-beta, Ltd.) is added, and 7 equivalents of TCEP per antibody molecule are added. Reduce at room temperature for 1 hour. The antibody reduction intermediate can be monitored by CE-SDS, and after the antibody is fully reduced, 20 mM linker-drug solution dissolved in DMSO is added, and 10 equivalents of linker-drug per antibody are added. React at 10°C for 1 hour. After the coupling is complete, the residual linker-drug is removed from the solution by ultrafiltration centrifuge tube (Merck & Co., Inc), and stored in PBS solution at pH 7.4. After purification, the average drug loading of the antibody in the ADC sample after coupling is detected by ultraviolet spectrophotometry.

[0214] Change the 5 mM TCEP equivalent per antibody molecule to 5, 3, and the 7, 5 equivalents of linker toxin per antibody molecule, and the average drug loading of the antibody in the ADC sample after coupling is detected by ultraviolet spectrophotometry.

[0215] The antibody sample is replaced by Trastuzumab (targeting HER2), 7 equivalents of TCEP per antibody molecule, and 10 equivalents of linker-drug. Using a similar method, the average drug loading of the antibody in the ADC sample after coupling is detected by ultraviolet spectrophotometry.

[0216] The antibody sample was replaced with SYM026(CDCP1), with each antibody molecule corresponding to 7 equivalents of TCEP and 10 equivalents of the linker-drug. Using a similar method, the average drug loading of the resulting ADC samples was determined by UV spectrophotometry. The 5mM TCEP equivalents corresponding to each antibody molecule were changed to 5 and 3, with each antibody molecule corresponding to 7 and 5 equivalents of the linker toxin. The average drug loading of the resulting ADC samples was then determined by UV spectrophotometry using the same method described above.

[0217] The statistical data of all ADC samples are shown in the table below.

[0218] Table 1.

[0219] Example 12: In vitro cell-killing activity test of ADC samples

[0220] Experimental materials:

[0221] HepG2 (human hepatocellular carcinoma, culture medium: MEM Medium + 10% FBS + 1% NEAA + 1mM NaP) and SKOV3 (human ovarian adenocarcinoma, culture medium: McCoy's 5a + 15% FBS + 1% P / S) cells were purchased from Nanjing Kebai Biotechnology Co., Ltd., and HuH7 (human hepatocellular carcinoma, culture medium: DMEM + 10% FBS) cells were donated by the Pharmacology Center of Shijiazhuang Pharmaceutical Group. All cells were obtained from ATCC (American Type Culture Collection).

[0222] Experimental methods:

[0223] Cell plating: When tumor cells reach a density of 80%-90%, digest the cells with trypsin (0.25% Trypsin-EDTA, Gibco, catalog number: 25200072), centrifuge at 1000 rpm for 5 min, resuspend the cells in complete culture medium and count them; take 75 μL of cell suspension and seed it into a black transparent bottom 96-well cell culture plate (Corning, catalog number: 3603), adjust the cell density, adjust the density of HepG2 and HuH7 cells to 3000 / well and the density of SKOV3 cells to 2250 / well, and incubate overnight in a CO2 cell culture incubator.

[0224] ADC sample preparation: Add complete medium and different samples to be tested in the first column of the U-bottom 96-well plate, 250ul / well; the concentration of the sample is 200nM; add complete medium to the second to eleventh columns, 200ul / well; take 50ul from the first column of samples and add to the second column, mix well, take 50ul from the second column of samples and add to the third column, and so on, 5-fold dilution to the ninth column.

[0225] Sample addition: The next day, after the cells adhered and grew, the prepared ADC samples of different concentrations were added to the 96-well cell culture plate, 75ul / well, 2 replicates for each sample, and incubated in a 37℃, 5% CO2 incubator for 6 days.

[0226] CellTiter-Lumi color development and plate reading: Add 50ul / well of CellTiter-Lumi reagent to the 96-well plate, and place it on a shaker at 300rpm in the dark for 10min to fully lyse the cells. After sufficient reaction, measure the chemiluminescence intensity on a multifunctional enzyme labeler. TM The CellTiter-Lumi cell viability detection reagent (item number: C0065XL) was equilibrated to room temperature in the dark, then 50uL of CellTiter-Lumi cell viability detection reagent was added to each well of the 96-well plate, and placed on a shaker at 300rpm in the dark for 10min to fully lyse the cells. After sufficient reaction, measure the chemiluminescence intensity on a multifunctional enzyme labeler.

[0227] MTT color development and plate reading: Dilute the dissolved MTT reagent with complete medium to 0.5mg / ml, and aspirate the medium in the 96-well cell culture plate, add complete medium containing 0.5mg / ml MTT reagent, 100ul / well, incubate in a 37℃, 5% CO2 incubator for 4 hours, then aspirate the medium, add DMSO, 150ul / well; and place it on a shaker at 300rpm in the dark for 10min, measure the OD value at 490nM with an enzyme labeler.

[0228] Data analysis: Process and analyze the data with Graphpad Prism 8.0, make a cell viability curve, and calculate the IC50 value. The results are shown in Tables 2 and 3:

[0229] Table 2.

[0230] Table 3.

[0231] Example 13: In vivo anti-tumor activity test of ADC samples

[0232] 1. In vivo efficacy of HepG2:

[0233] NCG mice were inoculated with human liver cancer HepG2 cells in the subcutaneous tissue of the axillary region of the forelimb, and when the tumor volume grew to about 130mm 3At the time, the animals were evenly grouped according to tumor volume, with 5-6 animals in each group, and were respectively given solvent, ADC2 3 mg / kg, and Lenvatinib 15 mg / kg. The solvent control group and ADC2 3 mg / kg were both administered by intravenous injection qw (Day 0 and Day 7); Lenvatinib 15 mg / kg was administered by gavage every day; and the amount of administration was 10 mL / kg.

[0234] The tumor diameters were measured twice a week, and the mouse body weight was measured, and the data were recorded. The growth changes of the tumors were dynamically observed by measuring the tumor diameters at different times after administration. After the end of the experiment (Day 18), the tumors were stripped and weighed to investigate the growth inhibition effect of ADC2 on human liver cancer HepG2 transplanted tumors. The data were analyzed by t-test, *p<0.05, **p<0.01, ***p<0.001, compared with the solvent group; #p<0.05, ##p<0.01, ###p<0.001, compared with the Lenvatinib group.

[0235] Tumor volume = V = 1 / 2 × a × b 2 (a and b represent the long and short diameters of the tumor, respectively)

[0236] Tumor weight inhibition (TWI) % = (1 - tumor weight of the administration group / tumor weight of the solvent group) × 100%

[0237] Test results

[0238] Under the test conditions, the mice in each group tolerated well, and no death occurred. Compared with the solvent control group, ADC2 and Lenvatinib had no significant change in the growth rate of the animal body weight, and the results are shown in Figure 1, left.

[0239] Compared with the solvent control group, ADC2 and Lenvatinib could significantly inhibit the growth of human liver cancer HepG2 tumors, and the tumor weight inhibition rates were 85.2% and 60.6%, respectively. ADC2 was significantly better than Lenvatinib (p<0.001), and details are shown in Table 4 and Figure 1, right.

[0240] Table 4 In vivo efficacy of ADC on human liver cancer HepG2 transplanted tumors

[0241] 2. In vivo efficacy of Huh-7

[0242] The NCG mice were inoculated with human liver cancer Huh-7 cells in the subcutaneous tissue of the axillary of the forelimb, and when the tumor volume grew to about 135 mm 3At the time, the animals were evenly grouped according to tumor volume, 7 animals in each group, and were respectively given solvent, ADC 25 mg / kg, and Lenvatinib 15 mg / kg. The solvent control group and ADC 25 mg / kg were both administered by single intravenous injection; Lenvatinib 15 mg / kg was administered by gavage every day.

[0243] The tumor diameters were measured twice a week, and the mouse body weight was measured and recorded. The growth changes of the tumors were dynamically observed by measuring the tumor diameters at different times after administration. After the end of the experiment (Day 13), the tumors were stripped and weighed to investigate the inhibitory effect of ADC2 on human hepatoma Huh-7 transplanted tumors. The data were analyzed by t-test, and compared with the solvent group, *p<0.05, **p<0.01, ***p<0.001; compared with the Lenvatinib group, #p<0.05, ##p<0.01, ###p<0.001.

[0244] Tumor volume = V = 1 / 2 x a x b2 (a and b represent the long and short diameters of the tumor, respectively)

[0245] Tumor weight inhibition (TWI) % = (1 - tumor weight of the administration group / tumor weight of the solvent group) x 100%

[0246] Test results

[0247] Under the conditions of this test, the mice in each group tolerated well and no death occurred. Compared with the solvent control group, ADC2 and Lenvatinib had no significant change in the growth rate of animal body weight, and the results are shown in Figure 2, left.

[0248] Compared with the solvent control group, ADC2 and Lenvatinib could significantly inhibit the growth of human hepatoma Huh-7 tumors, and the tumor weight inhibition rates were 96.1% and 69.3%, respectively. ADC2 was significantly better than Lenvatinib (p<0.001), and details are shown in Table 5 and Figure 2, right.

[0249] Table 5 In vivo efficacy of ADC on Huh-7 human hepatoma transplanted tumors

[0250] 3. In vivo efficacy of Hep3B

[0251] NCG mice were inoculated with human hepatoma Hep3B cells in the subcutaneous tissue of the axillary of the forelimb, and when the tumor volume grew to about 135 mm 3Animals were divided into equal groups of 6 animals each according to tumor volume. The groups were administered solvent, ADC2 4 mg / kg, and Lenvatinib 15 mg / kg, respectively. The solvent control group and ADC2 4 mg / kg were administered by single intravenous injection; Lenvatinib 15 mg / kg was administered by gavage daily.

[0252] Tumor diameter was measured twice weekly, and mouse weight was also recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after drug administration. At the end of the experiment (Day 17), tumors were removed and weighed to investigate the inhibitory effect of ADC2 on Hep3B hepatocellular carcinoma xenografts. Data were analyzed using t-tests; *p<0.05, **p<0.01, ***p<0.001.

[0253] Tumor volume = V = 1 / 2 × a × b² (where a and b represent the major and minor axes of the tumor, respectively)

[0254] Tumor weight inhibition rate (TWI)% = (1 - tumor weight in the drug-treated group / tumor weight in the solvent-treated group) × 100%

[0255] Test results

[0256] Under the conditions of this experiment, mice in the solvent group and ADC2 group tolerated the treatment well, and no deaths occurred. In the Lenvatinib group, one mouse died on Day 11 and Day 14, respectively. Compared with the solvent control group, ADC2 did not significantly change the rate of increase in animal weight, while Lenvatinib caused a significant decrease in the rate of increase in animal weight. The results are shown in the left figure of Figure 3.

[0257] Compared with the solvent control group, both ADC2 and Lenvatinib significantly inhibited the growth of human hepatocellular carcinoma Hep3B tumors, with tumor weight inhibition rates of 80.2% and 69.4%, respectively. ADC2 was superior to Lenvatinib; see Table 6 and the right side of Figure 3 for details.

[0258] Table 6. In vivo efficacy of ADCs against Hep3B-positive human liver cancer xenografts

[0259] 4. In vivo efficacy of SK-OV-3:

[0260] Human ovarian cancer SK-OV-3 cells were inoculated subcutaneously into the forelimb axilla of NU / NU mice until the tumor volume reached approximately 90 mm. 3 Animals were divided into equal groups according to tumor volume, with 5 animals in each group, and were given a single intravenous administration of solvent, ADC20 and ADC21 at 3 mg / kg.

[0261] The tumor diameters were measured twice a week, and the body weights of the mice were measured and recorded. The growth of the tumors was dynamically observed by measuring the tumor diameters at different times after administration. After the experiment, the tumors were stripped and weighed to investigate the inhibitory effects of ADC20 and ADC21 on the human ovarian cancer SK-OV-3 transplanted tumors.

[0262] Test results

[0263] Under the test conditions, the mice in each group tolerated well. ADC20 and ADC21 did not significantly change the growth rate of the animals compared with the solvent control group, and the results are shown in Fig. 4, left panel.

[0264] Compared with the solvent control group, ADC20 and ADC21 could significantly inhibit the growth of human ovarian cancer SK-OV-3 tumors, and the tumor weight inhibition rates were 81.8% and 89.8%, respectively. Details are shown in Table 7 and Fig. 4, right panel.

[0265] Table 7 In vivo efficacy of ADC on SK-OV-3 human ovarian cancer transplanted tumors

[0266] The above describes embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. An antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof: wherein Ab is selected from an antibody or an antigen binding fragment; Ab-(Z-W-D) m (I) Z is a linker unit; D is a drug unit; X is a connecting unit; W is represented by the formula: HG is a hydrophilic unit; PG is a protecting group; P is an enzyme cleavable unit; Y is a self-immolative unit; a end is connected to the linker unit Z, b end is connected to the drug unit D, wherein m is an integer or a decimal number from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); Preferably, wherein a1 end represents the connection to Ab, b1 end represents the connection to W; The linker unit Z is selected from or each of the alkyl, cycloalkyl, aryl, heteroalkyl, heteroaryl, heterocyclyl is optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino, carboxyl, or C3-C8 cycloalkyl; each of the heteroalkyl, heteroaryl, heterocyclyl contains 1-12 (preferably 1-8, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms selected from one or more of N, O, or S; and “multiple” means 2-12, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. U is a spacer unit selected from -U1-U2-U3-C(O)-, where U1 is selected from -C1-C8 alkyl-, -C1-C8 alkyl-C3-C8 cycloalkyl-, -C6-C 12 Aryl-, -C6-C 12 Aryl-C1-C8 alkyl-, -5-6-membered heteroaryl-, -5-6-membered heteroaryl-C1-C8 alkyl-, straight-chain or branched heteroalkyl groups with 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms, straight-chain or branched heteroalkyl-3-8-membered heterocyclic groups with 1-50 (preferably 1-20, more preferably 1-12, most preferably 1-8) carbon atoms; U2 is selected from bonds, -C 1-3 Alkyl-O-, -OC 1-3 Alkyl-, -O-, -C(O)-, -NR 1 -、-NR 1 -C(O)-、-C 1-3 Alkyl-NR 1 -or C 1-3 Alkyl-NR 1 -C(O)-,R 1 It is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic group, 2-8 membered heteroalkyl; U3 is selected from bond, C1-C8 alkyl, straight-chain or branched heteroalkyl with 2-10 (preferably 2-6) carbon atoms; The linker unit Z is selected from:

2. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically labeled versions thereof of claim 1, wherein, Preferably, U1 is selected from -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, phenylene, U2 is selected from a bond, -CH2NHC(O)-, -NHC(O)-, -O-, -CH2O-, -OCH2-, U3 is selected from a bond, -CH2OCH2-, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-; or Where a1 represents the link to the thiol group of the antibody, and b1 represents the link to the W group; U1 is selected from -C1-C6 alkyl-, -C6-C 12 aryl-, -C1-C3 alkyl-, -C3-C6 cycloalkyl, straight-chain or branched heteroalkyl with 1-20 carbon atoms; U2 is selected from bonds, -C 1-3 Alkyl-O-, -OC 1-3 Alkyl-, -O-, -C(O)-, -NR 1 -、-NR 1 -C(O)-、-C 1-3 Alkyl-NR 1 -or C 1-3 Alkyl-NR 1 -C(O)-,R 1 Selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclic, 2-6 membered heteroalkyl; U3 is selected from bond, C1-C8 alkyl, C 1-3 Alkyl-OC 1-3 Alkyl groups, wherein each of the alkyl and heteroalkyl groups is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, amino or carboxyl groups; wherein a1 end represents the connection to the thiol of the antibody, b1 end represents the connection to W. More preferably, U1is selected from -(CH2)5-, - U2-U3- is selected from the group consisting of: a bond, 3. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of claim 1 or 2, wherein, The linker unit Z is selected from the following structures: The connecting unit X of the linker unit is selected from:

4. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-3, wherein, wherein a4 end is connected to the enzyme cleavable unit P, b4 end is connected to the connecting unit X, c2 end is connected to the drug unit D. or X1is selected from -C(O)- or -NR 3 -, k and j each independently an integer from 0 to 10 (preferably 0 to 6, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), wherein a3denotes attachment to Z, b3denotes attachment to the self-immolition unit Y, and c1denotes attachment to the hydrophilic unit HG; R 2 and R 3 each independently selected from hydrogen, C1-C6alkyl, C3-C8cycloalkyl, 3-8 membered heterocyclyl, 2-8 membered heteroalkyl, each of said alkyl, heteroalkyl optionally substituted with one or more substituents independently selected from C1-C6alkyl, C1-C6alkoxy, amino, or carboxyl, said heterocyclyl, heteroalkyl containing 1-8 (such as 1, 2, 3, 4, 5, 6, 7, 8) heteroatoms selected from one or more of N, O, or S; said "one or more" in the foregoing definition is 2-8 (such as 1, 2, 3, 4, 5, 6, 7, 8) unless otherwise defined; Preferably, the linking unit X has preferably the following structure: More preferably, the linking unit X has preferably the following structure:

5. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-4, wherein, The self-cleaving unit Y is selected from the following structures: The enzyme cleavable unit P comprises an enzyme cleavable polypeptide connected to the self-immolative unit Y via an amide bond, wherein the polypeptide comprises a dipeptide having the sequence -P2-P1-, a tripeptide -P3-P2-P1-, a tetrapeptide -P4-P3-P2-P1-, a pentapeptide -P5-P4-P3-P2-P1-, and a hexapeptide -P6-P5-P4-P3-P2-P1-, wherein each of P1, P2, P3, P4, P5, and P6 is a natural amino acid or a non-natural amino acid; 6. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-5, wherein, Preferably, each of P1, P2, P3, P4, P5 is independently selected from alanine (A), asparagine (N), aspartic acid (D), glutamic acid (E), glycine (G), isoleucine (I), leucine (L), lysine (K), phenylalanine (F), proline (P), threonine (T), valine (V), and citrulline (C); ​ More preferably, each of P1, P2, P3, P4, P5 is independently selected from alanine (A), asparagine (N), glutamic acid (E), glycine (G), leucine (L), phenylalanine (F), valine (V), citrulline (C); Preferably, the enzyme cleavable unit P is selected from the following structures: wherein a5 is connected with a hydrophilic unit HG or a protecting group PG, and b5 is connected with a self-cleavage unit Y.

7. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-6, wherein, PG is selected from amino protecting groups, such as formyl, acetyl, trifluoroacetyl, benzoyl, alkoxycarbonyl (such as methoxycarbonyl, ethoxycarbonyl, allyloxycarbonyl, tert-butoxycarbonyl), benzyloxycarbonyl, fluorenylmethoxycarbonyl, and the like.

8. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-7, wherein, The enzyme cleavable unit P-PG is selected from the following structures: Preferably, the enzyme cleavable unit P-PG is:

9. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-8, wherein, The hydrophilic unit HG comprises polyethylene glycol, a peptide chain of natural or unnatural amino acids (such as polysarcosine), a sugar (such as monosaccharide, oligosaccharide, polysaccharide); Preferably, HG is selected from the following structures: wherein c3 is attached to a linking unit X or an enzyme cleavable unit P, S1 is selected from a bond, C1-C6 alkylene, 4-12 membered heteroalkylene, R 4 selected from hydrogen, C1-C6 alkyl, 2-6 membered heteroalkyl, HG1 is selected from a straight or branched chain heteroalkyl comprising 1-50 (preferably 6-24, more preferably 8-24) -OCH2CH2- structural units, or a straight or branched chain heteroalkyl comprising a 1-50 (preferably 6-24, more preferably 8-24) chain of natural or non-natural amino acids (such as a chain of -N(CH3)CH2C(O)- structural units), or a straight or branched chain heteroalkyl comprising a monosaccharide, oligosaccharide, or polysaccharide, each of said alkylene, heteroalkylene, alkyl, heteroalkyl being optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkoxy, amino, or carboxyl, said heteroalkylene, heteroalkyl containing 1-8 (such as 1, 2, 3, 4, 5, 6, 7, 8) heteroatoms selected from one or more of N, O, or S; said "plurality" in the foregoing definitions is 2-12 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) unless otherwise defined; Preferably, HG is selected from HG1 is selected from a straight-chain or branched heteroalkyl comprising 8-24 -OCH2CH2- structural units, or a straight-chain or branched heteroalkyl comprising 8-12 peptide chains of natural or unnatural amino acids (such as 8-12 -N(CH3)CH2C(O)- structural unit peptide chains), or a straight-chain or branched heteroalkyl comprising monosaccharide, oligosaccharide, or polysaccharide; Preferably, HG1is selected from -(CH2CH20)nCH3 n CH3, -(N(CH3)CH2C(O)) v OH, -CH2C(O)(N(CH3)CH2C(O)) v OH, wherein n and v are each independently selected from an integer of 1-50, preferably an integer of 1-30, further preferably an integer of 6-24, most preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30; More preferably, HG is selected from the following structures: Most preferably, HG is selected from the following structures:

10. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-9, wherein, -Z-W- is selected from the following structures: Preferably, -Z-W- is selected from the following structures: More preferably, -Z-W- is selected from the following structures: Most preferably, -Z-W- is selected from the following structures:

11. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-10, wherein, The drug unit D is selected from an amino-containing biologically active drug molecule or its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate, or isotope-labeled compound; Preferably, the drug unit D is selected from a camptothecin derivative, eribulin, an auristatin compound; More preferably, the drug unit D is selected from the group consisting of compounds of the following formulae: wherein c4 is connected with a self-cleavage unit Y; Most preferably, the active molecule D is selected from the group consisting of:

12. The antibody drug conjugate having the structure of Formula (I) of any one of claims 1-11 and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates, or isotopically-labeled forms thereof, selected from the following structures; Preferably, selected from the group consisting of: More preferably, selected from the group consisting of the following structures: Most preferably, selected from the following structures: wherein Ab is selected from an antibody or an antigen-binding fragment; m represents the molar ratio of the drug unit D to Ab (also referred to as DAR, i.e., drug antibody coupling ratio), which is an integer or a decimal number from 1 to 12, for example, an integer or a decimal number from 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; when m is a decimal number, it represents the average molar ratio of the drug unit D to Ab.

13. The antibody drug conjugate having the structure of Formula (I) and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopically-labeled forms thereof of any one of claims 1-12, wherein, The antibody moiety Ab is selected from a single antibody, a double antibody, or an antigen-binding fragment thereof targeting HER2 (ErbB2), GPC3, or CDCP1; Preferably, Ab is an antibody or an antigen-binding fragment thereof targeting GPC3, which comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), the heavy chain variable region comprising 3 complementarity determining regions (CDRs), wherein the heavy chain complementarity determining region 1 (HCDR1) amino acid sequence is shown as SEQ ID NO: 1, the heavy chain complementarity determining region 2 (HCDR2) amino acid sequence is shown as SEQ ID NO: 2, and the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence is shown as SEQ ID NO: 3; the light chain variable region comprising 3 complementarity determining regions (LCDRs), wherein the light chain complementarity determining region 1 (LCDR1) amino acid sequence is shown as SEQ ID NO: 4, the light chain complementarity determining region 2 (LCDR2) amino acid sequence is shown as SEQ ID NO: 5, and the light chain complementarity determining region 3 (LCDR3) amino acid sequence is shown as SEQ ID NO: 6, wherein the CDRs are determined according to the Kabat numbering rule; or Ab is an antibody or antigen-binding fragment thereof targeting HER2, comprising a heavy chain variable region comprising 3 complementarity determining regions (CDRs), wherein the heavy chain complementarity determining region 1 (HCDR1) amino acid sequence is set forth in SEQ ID NO: 11, the heavy chain complementarity determining region 2 (HCDR2) amino acid sequence is set forth in SEQ ID NO: 12, and the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence is set forth in SEQ ID NO: 13; and / or a light chain variable region comprising 3 complementarity determining regions (CDRs), wherein the light chain complementarity determining region 1 (LCDR1) amino acid sequence is set forth in SEQ ID NO: 14, the light chain complementarity determining region 2 (LCDR2) amino acid sequence is set forth in SEQ ID NO: 15, and the light chain complementarity determining region 3 (LCDR3) amino acid sequence is set forth in SEQ ID NO: 16, wherein the CDRs are determined according to the Kabat numbering convention; or Ab is an antibody or antigen-binding fragment thereof targeting CDCP1, comprising a heavy chain variable region comprising 3 complementarity determining regions (CDRs), wherein the heavy chain complementarity determining region 1 (HCDR1) amino acid sequence is set forth in SEQ ID NO: 21, the heavy chain complementarity determining region 2 (HCDR2) amino acid sequence is set forth in SEQ ID NO: 22, and the heavy chain complementarity determining region 3 (HCDR3) amino acid sequence is set forth in SEQ ID NO: 23; and / or a light chain variable region comprising 3 complementarity determining regions (CDRs), wherein the light chain complementarity determining region 1 (LCDR1) amino acid sequence is set forth in SEQ ID NO: 24, the light chain complementarity determining region 2 (LCDR2) amino acid sequence is set forth in SEQ ID NO: 25, and the light chain complementarity determining region 3 (LCDR3) amino acid sequence is set forth in SEQ ID NO: 26, wherein the CDRs are determined according to the Kabat numbering convention; More preferably, Ab is an antibody or antigen-binding fragment targeting GPC3, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or Ab is an antibody or antigen-binding fragment thereof targeting HER2, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18; or Ab is an antibody or antigen-binding fragment thereof targeting CDCP1, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 27 and / or a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 28; More preferably, Ab is an antibody targeting GPC3, which comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO: 9, and the amino acid sequence of the light chain is shown as SEQ ID NO: 10; or Ab is an antibody targeting HER2, which comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO: 19, and the amino acid sequence of the light chain is shown as SEQ ID NO: 20; or Ab is an antibody targeting CDCP1, which comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown as SEQ ID NO: 29, and the amino acid sequence of the light chain is shown as SEQ ID NO:

30.

14. A linker-drug compound represented by formula (II) and its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotope-labeled compound thereof: Z'-W-D (II) wherein Z' is a precursor of the linker unit capable of being converted into Z of formula (I) to form a covalent bond with Ab of formula (I); W, D and Ab are defined as in formula (I) of claim 1. Z'-W-D (II) wherein Z' is a precursor of the linker unit capable of being converted into Z of formula (I) to form a covalent bond with Ab of formula (I); W, D and Ab are defined as in formula (I) of claim 1. wherein U is defined as in formula (I) of claim 1.

16. A linker represented by formula (III) and its tautomer, stereoisomer, solvate or isotope-labeled compound thereof, selected from the following structures: Z'-W' (III) wherein Z' is a first precursor of the linker unit capable of being converted into Z of formula (I) to form a covalent bond with Ab of formula (I); and W' is a second precursor of the linker unit capable of being converted into W of formula (I) to form a covalent bond with D of formula (I); and Preferably, the linker unit precursor Z' is selected from or wherein the foregoing Z', Z, W, D and Ab are defined as in formula (I) of claim 1 or formula (II) of claim 14; More preferably, the linker unit Z' is selected from the following structures:

15. The linker-drug compound of formula (II) as shown in claim 14 and its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, solvates or isotopic labels, selected from the following structures: More preferably, selected from the group consisting of the following structures: Most preferably, R1is selected from the following structures: wherein the foregoing a, X, HG, PG, P, a4, b4 are defined as in formula (I) of any one of claims 1-5; 17. A pharmaceutical composition comprising an antibody-drug conjugate represented by formula (I) of any one of claims 1-13 and its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotope-labeled compound thereof, or a linker-drug having the structure of formula (II) of claim 14 or 15 and its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotope-labeled compound thereof, and optionally one or more pharmaceutically acceptable excipients.

18. Use of an antibody-drug conjugate represented by formula (I) of any one of claims 1-13 and its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotope-labeled compound thereof, or a linker-drug having the structure of formula (II) of claim 14 or 15 and its tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, solvate or isotope-labeled compound thereof, or a pharmaceutical composition of claim 17 in the manufacture of a medicament for the treatment of cancer. Preferably, W' is represented by the formula: wherein Y' is selected from the following structures: ​ More preferably, formula (III) is selected from the following structures: Most preferably, formula (III) is selected from the following structures: ​ ​ 19. The use according to claim 18, wherein the cancer comprises liver cancer, ovarian cancer, colorectal cancer, breast cancer.

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