Antibody drug conjugates

Abstract

The invention provides an antibody drug conjugate which specifically comprises a therapeutic antibody part, an intermediate joint part and a cytotoxic drug part which are connected, the therapeutic antibody part is an antibody aiming at an HER2 target spot, and the cytotoxic drug part is a camptothecin topoisomerase I inhibitor. The cytotoxic drug part or the linker-cytotoxic drug part is subjected to deuterium-substituted modification. The antibody drug conjugate can be used for preventing or treating cancers. # imgabs0 #

Description

[0001] This application is a divisional application of the invention patent application with the application date of August 13, 2021, Chinese application number 202180041403.9, and invention name “Antibody Drug Conjugate”. Technical Field

[0002] The present application relates to an antibody-drug conjugate, which comprises a therapeutic antibody portion, an intermediate linker portion and a cytotoxic drug portion connected to each other. The present application also relates to the use of the antibody-drug conjugate in preparing a drug for preventing and treating cancer. Background Art

[0003] Antibody-Drug Conjugate (ADC) is a class of drugs that combines the high specificity of therapeutic antibodies with the high killing activity of cytotoxic drugs, in which the therapeutic antibody part and the cytotoxic drug part are connected through the intermediate linker part. At present, at least eight ADC drugs have been launched globally, among which the antibody parts of brentuximab vedotin, polatuzumab vedotin and enfortumab vedotin target CD30, CD79b and Nectin-4 respectively, the antibody parts of trastuzumab emtansine and trastuzumab deruxtecan target HER2, the antibody parts of gemtuzumab ozogamicin and inotuzumab ozogamicin target CD33 and CD22 respectively, and the antibody part of sacituzumab govitecan targets TROP2. As the cytotoxic drug part, brentuximab vedotin, polatuzumab vedotin and enfortumab vedotin use auristatins toxin molecules that act on microtubules, trastuzumab emtansine uses maytansinoid toxin molecules that act on microtubules, gemtuzumab ozogamicin and inotuzumab ozogamicin use calicheamicins toxin molecules that act on DNA, and the latest trastuzumab deruxtecan and sacituzumab govitecan both use camptothecin toxin molecules. As the intermediate linker part, trastuzumabemtansine uses a non-cleavable linker, and the other seven ADC drugs mentioned above all use cleavable linkers.

[0004] Camptothecin (CPT) analogs and derivatives exert antitumor activity by binding to topoisomerase I, and they have shown significant activity against many tumor types. In order to overcome the defect of poor water solubility of CPT, researchers have synthesized a variety of CPT derivatives, among which irinotecan hydrochloride (CPT-11) is a water-soluble prodrug that has been approved for the treatment of metastatic colorectal cancer. However, CPT-11 must be catalyzed by carboxylesterase in the body to be converted into its active form SN-38 (Formula I), and this conversion efficiency is extremely low. SN38 itself is difficult to make into a drug due to its poor solubility. Exatecan (Formula II) (English generic name: exatecan) is another water-soluble CPT derivative that has been attempted to be developed as an antitumor drug, but development was stopped in 2004. It does not require activation by the use of enzymes. In addition, compared with SN-38, which is the pharmacological body of irinotecan, exatecan has stronger topoisomerase I inhibitory activity.

[0005]

[0006] ADC drugs combine the dual advantages of the high efficacy of cytotoxic small molecules and the high selectivity of antibodies for specific tumor cells. There is still a need to develop highly effective and low-toxic ADC drugs that can target more indications. Summary of the invention

[0007] In one aspect, the present application provides a deuterated modified antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and specifically relates to a deuterated modification of a linker or a cytotoxic drug portion.

[0008] In one aspect, the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody portion, L represents a linker portion, U represents a cytotoxic drug portion, and n is an integer or decimal selected from 1 to 10.

[0009] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab (antibody portion) can specifically bind to a tumor antigen (including a tumor-specific antigen and a tumor-associated antigen), and the tumor antigen can be selected from any tumor prevention or treatment target known in the art, for example, can be selected from HER2, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1, PD-L1, etc.

[0010] In some embodiments, the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab (antibody portion) can be modified, for example, including the change, addition or reduction of one or more amino acids.

[0011] In some embodiments, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab is an antibody that can specifically bind to HER2.

[0012] In some embodiments, the antibody portion Ab of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is trastuzumab, which has the sequence shown in Table S1 below.

[0013] Table S1 Trastuzumab sequence

[0014]

[0015] In some embodiments, the antibody portion Ab of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is Pertuzumab, which has the sequence shown in Table S2 below.

[0016] Table S2 Pertuzumab sequence

[0017]

[0018] In some embodiments, the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a first antigen binding fragment that monovalently and specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen binding fragment being a scFv comprising VH and VL, the VH having a K30 mutation, and / or the VL having a F53 mutation. In some embodiments, the 30th amino acid in the sequence of the VH mutates from K to an acidic amino acid, such as E. In some embodiments, the 53rd amino acid in the sequence of the VL mutates from F to a neutral amino acid or a basic amino acid, such as Y, A, R. For example, the scFv may comprise or be a sequence having a K30 mutation and / or a F53 mutation in the amino acid sequence shown in SEQ ID NO: 1.

[0019] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment being a scFv, the first antigen-binding fragment comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 respectively comprising the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29, the light chain CDR1, the light chain CDR2 and the light chain CDR3 respectively comprising the amino acid sequences shown in SEQ ID NOs: 30, 34 and 32; wherein the sequence shown in SEQ ID NO: 27 is GNFIX2DTYIH, and X2 is K or E; the sequence shown in SEQ ID NO: 34 is SASX1LYS, and X1 is F or Y.

[0020] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment is a scFv, the first antigen-binding fragment comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 43, 28 and 29, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 30, 31 and 32.

[0021] In one aspect, the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a first antigen binding fragment that monovalently and specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, wherein the first antigen binding fragment is a scFv and is selected from:

[0022] i. the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 41 and 42, respectively; or

[0023] ii. the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 41 and 42;

[0024] The sequence shown in SEQ ID NO: 41 is:

[0025] EVQLVESGGGLVQPGGSLRLSCAASGFNIX2DTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, X2 is K or E; the sequence shown in SEQ ID NO: 42 is:

[0026] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASX1LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, X1 is F or Y.

[0027] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment is a scFv, and the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 35 and 36.

[0028] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a first antigen-binding fragment that monovalently and specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment is a scFv, and the VH and VL of the first antigen-binding fragment are arranged in the following order from N-terminus to C-terminus: VH-linker-VL.

[0029] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab further comprises a second antigen-binding fragment that monovalently and specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, and the second antigen-binding fragment is Fab.

[0030] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab further comprises a second antigen-binding fragment that monovalently and specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, the second antigen-binding fragment being Fab and comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 45, 46 and 47, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprising the amino acid sequences shown in SEQ ID NOs: 48, 49 and 50, respectively.

[0031] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab further comprises a second antigen-binding fragment that monovalently and specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, wherein the second antigen-binding fragment is Fab and comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise the amino acid sequences shown in SEQ ID NOs: 37 and 38, respectively.

[0032] In some specific embodiments, the antibody portion Ab of the antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is as shown in Table S3.

[0033] Table S3 Antibody portion of exemplary antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof

[0034]

[0035] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, and the immunoglobulin functional domain comprises: i. CL, CH1, CH2, or CH3, or ii. Fc.

[0036] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, wherein the immunoglobulin functional domain comprises: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc, wherein the CL, CH1, CH2, CH3, and Fc are derived from the CL, CH1, CH2, CH3, and Fc of human IgG, respectively.

[0037] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises an immunoglobulin domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, wherein the immunoglobulin domain comprises: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc, wherein the CL, CH1, CH2, CH3 or Fc has a modification or has no modification; preferably, the modification of the CH3 or Fc is, for example, a substitution of the 435th and / or 436th amino acid according to the Kabat numbering system.

[0038] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, the immunoglobulin functional domain comprising: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc, wherein the Fc is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, the first antigen-binding fragment is operably linked to the first Fc polypeptide, and the second antigen-binding fragment is operably linked to the second Fc polypeptide.

[0039] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a constant region operably linked to the first antigen-binding fragment and / or the second antigen-binding fragment, and the constant region can be a native sequence constant region of an immunoglobulin or a mutated constant region, such as one or more of the native or mutated CL, CH1, CH2, and / or CH3 functional domains. In some examples, these functional domains are operably linked by conventional means in the art, and the constant region can be derived from the constant region of a human immunoglobulin, such as from IgG1, IgG2, IgG3 or IgG4. In some examples, the constant region can be modified to improve its ability to mediate effector functions.

[0040] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises an immunoglobulin functional domain or skeleton, such as Fc, which is operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment. The term Fc includes native sequence Fc regions and variant Fc regions. Fc can be human Fc such as derived from IgG1, IgG2, IgG3 or IgG4. Fc can have modifications to improve its ability to mediate effector function. For example, in some embodiments, the skeleton has modifications such as knob into hole, H435R, Y436F, de-fucose, etc. In some embodiments, the mutation sites of the knob into hole of the above-mentioned skeleton include, for example, Y349C, T366S, L368A, Y407V, S354C and T366W, etc.

[0041] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises a skeleton operably connected to a first antigen-binding fragment and / or a second antigen-binding fragment; in some embodiments, the skeleton is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide; in some embodiments, the dimeric Fc has a modification; in some embodiments, the dimeric Fc has H435R and / or Y436F, and the modification can be in any one of the first Fc polypeptide and the second Fc polypeptide. chain; in some specific embodiments, the dimeric Fc has H435R and / or Y436F, and the modification occurs only on one Fc polypeptide but not on the other Fc polypeptide; in some embodiments, the dimeric Fc has a knob-into-hole mutation site, such as Y349C, T366S, L368A, Y407V, S354C and T366W, etc.; in some embodiments, one chain of the dimeric Fc has T366W and / or S354C, and the other chain has Y407V, Y349C, T366S or / and L368A.

[0042] In one aspect, the present application provides an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab is a bivalent bispecific antibody comprising: a heavy chain comprising the sequence of SEQ ID NO: 11, a heavy chain comprising the sequence of SEQ ID NO: 13, and a light chain comprising the sequence of SEQ ID NO: 15.

[0043] In some specific embodiments, the antibody Ab portion of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is as shown in Table S4.

[0044] Table S4 Antibody portion of exemplary antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof

[0045]

[0046]

[0047]

[0048] In one aspect, the present application provides an antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug portion U is coupled to the antibody portion Ab via a linker portion L. The linker portion L of the present application can be connected to the antibody portion by any method known in the art, preferably the linker portion is connected to the antibody portion via a sulfhydryl group and / or an amino group. In some more preferred embodiments, the linker portion of the present application is connected to the antibody portion via a sulfhydryl group.

[0049] In one aspect, the present application provides an antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug portion U is coupled to the antibody portion Ab via a linker portion L, and the linker portion may be a cleavable linker or a non-cleavable linker; in some embodiments, the linker portion of the present application is a cleavable linker, for example, it may be a low pH-dependent degradation type (including hydrazone bonds, carbonate bonds, etc.), a proteolytic type (including peptide bonds), or a high glutathione concentration-dependent degradation type (including disulfide bonds), etc.; in other embodiments, the linker portion of the present application is a non-cleavable linker, for example, it may be a maleimidocaproyl group, etc.

[0050] In one aspect, the present application provides an antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab is coupled to one or more cytotoxic drug portions U, and the cytotoxic drug can be selected from, for example, alkaloids, antimetabolites, antitumor antibiotics, alkylating agents and platinums, etc., and the preferred cytotoxic drug is a microtubule inhibitor cytotoxic drug (including maytansines, auristatins) or a cytotoxic drug acting on DNA (including calicheamicins, duocarmycins, PBD (pyrrolobenzodiazepine) class, topoisomerase I inhibitors, etc.).

[0051] In some specific embodiments, the cytotoxic drug portion U of the antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is a topoisomerase I inhibitor.

[0052] In some specific embodiments, the cytotoxic drug portion U of the antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is a camptothecin topoisomerase I inhibitor.

[0053] In some preferred embodiments, the cytotoxic drug portion U of the antibody-drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is selected from SN-38, SN-38 derivatives, exatecan or exatecan derivatives.

[0054] In some embodiments, the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody portion, L represents a linker portion, U represents a camptothecin-type topoisomerase I inhibitor, and n is an integer or decimal selected from 1 to 10, wherein the L portion and / or the U portion are deuterated. In some embodiments, n is an integer or decimal selected from 2 to 10, such as an integer or decimal of 2 to 9, an integer or decimal of 2 to 8, an integer or decimal of 3 to 9, an integer or decimal of 3 to 8, an integer or decimal of 4 to 9, an integer or decimal of 4 to 8, an integer or decimal of 5 to 9, or an integer or decimal of 5 to 8.

[0055] In some embodiments, the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody portion, L represents a linker portion, U represents a camptothecin-type topoisomerase I inhibitor, and n is an integer or decimal selected from 1 to 10, wherein the L portion and / or the U portion is deuterated, and the cytotoxic drug portion U is selected from SN-38, an SN-38 derivative, exatecan, or an exatecan derivative.

[0056] In some embodiments, the antibody drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula III:

[0057]

[0058] In some embodiments, the antibody drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula IV:

[0059]

[0060] Wherein, R1 is selected from hydrogen (H) or deuterium (D).

[0061] In some embodiments, the antibody drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula IV-1 or IV-2:

[0062]

[0063] In some embodiments, the antibody drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula V:

[0064]

[0065] Wherein, R1 and R2 are independently selected from hydrogen (H) or deuterium (D),

[0066] Furthermore, the left succinimide terminal of the structure is the linking site to the antibody portion, and the right carbonyl terminal is the linking site to the cytotoxic drug portion.

[0067] In some embodiments, the antibody-drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula V-1, V-2, V-3 or V-4, and the structures shown in V-1 to V-4 are respectively connected to the antibody portion at the left succinimide terminal and connected to the cytotoxic drug portion at the right carbonyl terminal:

[0068]

[0069] In some embodiments, the antibody drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula VI:

[0070]

[0071] in,

[0072] R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0073] In some embodiments, the antibody drug conjugate of the general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown in the following formula VI-1, VI-2, VI-3 or VI-4:

[0074]

[0075]

[0076] In some specific embodiments, the present application provides an antibody drug conjugate having a structure shown in Formula VII or a pharmaceutically acceptable salt or solvate thereof:

[0077]

[0078] in,

[0079] Ab represents an antibody portion, which comprises a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment is a scFv and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 30, 31 and 32, respectively;

[0080] The second antigen-binding fragment is a Fab and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 45, 46 and 47, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 48, 49 and 50, respectively,

[0081] n is an integer or decimal selected from 1 to 10,

[0082] R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0083] In some specific embodiments, the present application provides an antibody drug conjugate having a structure shown in the following formula VII-1, VII-2, VII-3 or VII-4, or a pharmaceutically acceptable salt or solvate thereof,

[0084]

[0085]

[0086] in,

[0087] Ab represents an antibody portion, which comprises a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment is a scFv and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 30, 31 and 32, respectively;

[0088] The second antigen-binding fragment is a Fab and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 45, 46 and 47, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 48, 49 and 50, respectively,

[0089] n is an integer or a decimal selected from 1 to 10.

[0090] In a specific embodiment, the present application provides an antibody drug conjugate having a structure shown in Formula VII-1 or a pharmaceutically acceptable salt or solvate thereof,

[0091]

[0092] in,

[0093] Ab represents an antibody portion, which comprises a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment is a scFv and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 30, 31 and 32, respectively;

[0094] The second antigen-binding fragment is a Fab and comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 45, 46 and 47, respectively, and the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 48, 49 and 50, respectively,

[0095] n is an integer or a decimal selected from 1 to 10.

[0096] In a specific embodiment, the present application provides an antibody drug conjugate having a structure as shown in Formula VII or a pharmaceutically acceptable salt or solvate thereof,

[0097]

[0098] in,

[0099] Ab is trastuzumab,

[0100] n is an integer or decimal selected from 1 to 10,

[0101] R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0102] In a specific embodiment, the present application provides an antibody drug conjugate having a structure shown in Formula VII-1 or a pharmaceutically acceptable salt or solvate thereof,

[0103]

[0104] in,

[0105] Ab is trastuzumab,

[0106] n is an integer or a decimal selected from 1 to 10.

[0107] In one aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate according to the present application or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0108] In one aspect, the present application provides use of the antibody-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for preventing or treating cancer.

[0109] In one aspect, the present application provides use of a pharmaceutical composition comprising the antibody-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for preventing or treating cancer.

[0110] In one aspect, the present application provides an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for preventing or treating cancer.

[0111] In one aspect, the present application provides a method for treating or preventing cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the antibody-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

[0112] In some embodiments, the antibody-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat HER2-positive cancer, HER2-negative cancer (including triple-negative breast cancer), and cancers that show HER2 expression as IHC2+ according to immunohistochemistry detection.

[0113] In some aspects, the present application provides a linker-drug intermediate compound having a structure shown in the following formula VI, which is used to obtain an antibody-drug conjugate formed by connecting an antibody to the intermediate compound:

[0114]

[0115] in,

[0116] R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0117] In some aspects, the present application provides a linker-drug intermediate compound having a structure shown in the following formula VI-1, VI-2, VI-3 or VI-4, which is used to obtain an antibody-drug conjugate formed by connecting an antibody to the intermediate compound:

[0118]

[0119]

[0120] In some aspects, the present application provides a linker compound having a structure shown in the following formula V, which is used to obtain an antibody-drug conjugate formed by connecting a drug to an antibody via the linker:

[0121]

[0122] Wherein, R1 and R2 are independently selected from hydrogen (H) or deuterium (D),

[0123] Furthermore, the left succinimide terminal of the structure is the linking site to the antibody portion, and the right carbonyl terminal is the linking site to the cytotoxic drug portion.

[0124] In some aspects, the present application provides a linker compound having a structure shown in the following formula V-1, V-2, V-3 or V-4, which is used to obtain an antibody-drug conjugate formed by connecting a drug to an antibody via the linker, and the structures shown in formulas V-1 to V-4 are respectively connected to the antibody portion at the left succinimide terminal and connected to the cytotoxic drug portion at the right carbonyl terminal:

[0125]

[0126]

[0127] In some aspects, the present application provides a compound having a structure shown in the following formula IV (a):

[0128]

[0129] Wherein, R1 is selected from hydrogen (H) or deuterium (D).

[0130] In some aspects, the present application provides a compound having a structure shown in the following formula IV(a)-1 or IV(a)-2:

[0131]

[0132] In some aspects, the present application provides a compound having a structure shown in the following formula III(a):

[0133]

[0134] The present application provides an antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof with improved pharmacokinetic properties. The improved pharmacokinetic properties will lead to reduced toxicity of the target compound, improved safety and / or tolerability, improved efficacy, and ultimately improved therapeutic window. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 The killing rate of anti-HER2 bispecific antibodies (Expi HER2-1, Expi HER2-2, 23C2 HER2-1 and 23C2HER2-2) and the combination of trastuzumab + pertuzumab on BT474 tumor cells;

[0136] Figure 2 The killing rate of anti-HER2 bispecific antibody, trastuzumab, T-DM1, and the combination of trastuzumab + pertuzumab on NCI-N87 tumor cells;

[0137] Figure 3The killing rate of anti-HER2 bispecific antibody, trastuzumab, T-DM1, and the combination of trastuzumab + pertuzumab on JIMT-1 tumor cells;

[0138] Figure 4 The results of the proliferation inhibition of BT474 tumor cells by anti-HER2 bispecific antibody, trastuzumab, and the combination of trastuzumab + pertuzumab;

[0139] Figure 5 The effects of anti-HER2 bispecific antibody, PBS vehicle control, and combined trastuzumab + pertuzumab (Per+Tra) on the changes in tumor volume in the gastric cancer N87 mouse xenograft model;

[0140] Figure 6 The effects of anti-HER2 bispecific antibody, PBS vehicle control, and combined trastuzumab + pertuzumab on the weight changes of mice during the drug efficacy of gastric cancer N87 mouse xenograft tumors;

[0141] Figure 7 The structures of some exemplary anti-HER2 bispecific antibodies; wherein a dimeric Fc is depicted with one chain shown in black (the first Fc polypeptide) and another chain shown in gray (the second Fc polypeptide), and one antigen binding domain (the first antigen binding fragment) is shown as hatched, and the other antigen binding domain (the second antigen binding fragment) is shown in white; the first antigen binding fragment is a scFv fused to the first Fc polypeptide, and the second antigen binding fragment is a Fab fused to the second Fc polypeptide;

[0142] Figure 8 The endocytic activity of different antibody-drug conjugates (monoclonal antibody-DDDXD and bispecific antibody-DDDXD) in NCI-N87 tumor cells;

[0143] Fig. 9 The endocytic activity of different antibody-drug conjugates (monocytic antibody-DDDXD and bispecific antibody-DDDXD) in SK-BR-3 tumor cells.

[0144] Explanation and Definitions

[0145] Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered as uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (2012); Abbas et al., Cellular and Molecular Immunology, Elsevier Science Health Science div (2009); He Wei et al., Medical Immunology (2nd Edition), People's Medical Publishing House, 2010. When a trade name appears in this article, it is intended to refer to the corresponding commodity or its active ingredient.

[0146] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxo will not occur on an aromatic group.

[0147] The term "optional" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes the occurrence of the event or situation and the non-occurrence of the event or situation. A certain group is "optionally substituted" means that the group may be substituted or unsubstituted, for example, an ethyl group is "optionally" substituted with a halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (such as CH2CH2F), polysubstituted (such as CHFCH2F, CH2CHF2, etc.) or completely substituted (CF2CF3). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that is sterically impossible to exist and / or cannot be synthesized will be introduced.

[0148] In this article, C m-n , means that the moiety has an integer number of carbon atoms in a given range. For example, "C 1-6” means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0149] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 2 R's, each R has an independent choice.

[0150] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a covalent bond.

[0151] When one of the variables is selected from a covalent bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a covalent bond, it means that the structure is actually AZ.

[0152] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0153] The term "hydroxy" refers to an -OH group.

[0154] The term "cyano" refers to a -CN group.

[0155] The term "mercapto" refers to a -SH group.

[0156] The term "amino" refers to a -NH2 group.

[0157] The term "nitro" refers to a -NO2 group.

[0158] The term "alkyl" refers to a group of the formula C n H 2n+1 The alkyl group may be straight chain or branched. For example, the term "C 1-6 The term "alkyl" refers to an alkyl group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio has the same definition as above.

[0159] The term "alkoxy" refers to an -O-alkyl group.

[0160] The term "alkylamino" refers to an -NH-alkyl group.

[0161] The term "dialkylamino" refers to -N(alkyl)2.

[0162] The term "alkylsulfonyl" refers to a -SO2-alkyl group.

[0163] The term "alkylthio" refers to an -S-alkyl group.

[0164] The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms having at least one double bond. Non-limiting examples of alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.

[0165] The term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms having at least one triple bond. Non-limiting examples of alkynyl include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH3), 2-propynyl (-CH2-C≡CH), 1,3-butadiynyl (-C≡CC≡CH), etc.

[0166] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is typically a 3 to 10 membered ring. Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, etc.

[0167] The term "cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and can exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is typically a 5 to 8 membered ring. Non-limiting examples of cycloalkenyl include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, etc.

[0168] The term "heterocyclic radical" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated heteroaromatic) and can exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the heterocycle is generally a 3 to 7 ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and / or nitrogen. Non-limiting examples of heterocyclic radicals include but are not limited to oxirane, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl etc.

[0169] The term "heterocycloalkyl" refers to a cyclic group that is fully saturated 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 (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and / or nitrogen. Examples of 3-membered heterocycloalkyls include, but are not limited to, oxirane, thioethane, and cyclonitroethane. Non-limiting examples of 4-membered heterocycloalkyls include, but are not limited to, azetidinyl, oxadiazolyl, and thiadinyl. Examples of 5-membered heterocycloalkyls include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, and tetrahydropyrazolyl. Examples of 6-membered heterocycloalkyls include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thioxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl, and 1,4-dithianyl. Examples of 7-membered heterocycloalkyls include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Monocyclic heterocycloalkyls having 5 or 6 ring atoms are preferred.

[0170] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. For example, an aryl group may have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and 1,2,3,4-tetrahydronaphthalene, etc.

[0171] The term "heteroaryl" refers to a monocyclic or fused polycyclic system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryls have a single 4 to 8 ring, especially a 5 to 8 ring, or a plurality of fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryls include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.

[0172] "Derivative": A compound formed by the replacement of atoms or atomic groups in the molecule of a parent compound by other atoms or atomic groups is called a derivative of the parent compound.

[0173] Any atom of the synthetic compound marked in this application, if not specifically specified, may represent any stable isotope of the atom. Unless otherwise specified, when a position in the structure is defined as H, i.e., hydrogen (H-1), the position contains only naturally occurring isotopes. Similarly, unless otherwise specified, when a position in the structure is defined as D, i.e., deuterium (H-2), the amount of isotopes contained in the position is at least 3340 times greater than the amount of naturally occurring isotopes (0.015%) (i.e., at least 50.1% deuterium isotopes). When one or more positions in the structure of the marked synthetic compound are defined as D, i.e., deuterium (H-2), the content of the compound shown in the structure may be at least 52.5%, at least 60%, at least 67.5%, at least 75%, at least 82.5%, at least 90%, at least 95%, at least 97%, at least 98.5%, at least 99%, at least 99.5%. The deuteration rate of the labeled synthetic compound in the present application refers to the ratio of the labeled synthetic isotope content to the amount of the naturally occurring isotope. The deuteration rate of each designated deuterium atom of the labeled synthetic compound in the present application may be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), at least 6600 times (99%), at least 6633.3 times (99.5%). Isotopologues in the present application refer to compounds that differ only in isotopic composition in terms of chemical structure. The compounds synthesized by marking in the present application have the same chemical structure, with only isotopic changes in the atomic composition of their molecules. Therefore, the deuterium-containing compounds synthesized by marking in the present application at a specific position will also contain very few hydrogen isotopologues at that position. The amount of hydrogen isotopologues at a certain position in the compounds synthesized by marking in the present application depends on many factors, including the deuterium isotopic purity of the deuterated reagent (D2O, D2, NaBD4, LiAlD4, etc.) and the effectiveness of the synthesis method for introducing deuterium isotopes. However, as mentioned above, the total amount of hydrogen isotopologues at such a certain position will be less than 49.9%. The total amount of hydrogen isotopologues at a certain position in the compounds synthesized by marking in the present application will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1% or 0.5%.

[0174] In this application, any atom not designated as deuterium is present at its natural isotopic abundance.

[0175] The term "treatment" means administering the compounds or preparations described herein to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) preventing a disease or disease state from occurring in a mammal, especially when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state; (ii) inhibiting a disease or disease state, i.e., curbing its development; and (iii) alleviating a disease or disease state, i.e., causing the disease or disease state to regress.

[0176] The term "therapeutically effective amount" means an amount of the compound of the present application that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of the compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0177] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0178] As the pharmaceutically acceptable salt, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned.

[0179] The term "solvate" refers to an association of a compound with solvent molecules.

[0180] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

[0181] The term "pharmaceutically acceptable excipients" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0182] The compounds and intermediates of the present application can also exist in different tautomeric forms, and all such forms are included in the scope of the present application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as prototransfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. The specific example of a proton tautomer is an imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions by the reorganization of some bonding electrons.

[0183] The term "antibody" is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (eg, bispecific antibodies), multifunctional antibodies, and antibody fragments, so long as they have the desired biological activity.

[0184] The term "humanized antibody" refers to an antibody comprising CDR regions derived from a non-human antibody, while the rest of the antibody molecule is derived from one or more human antibodies.

[0185] The term "mutant" is used to refer to a peptide comprising an amino acid sequence derived from the amino acid sequence of the peptide as follows: one or two or more amino acids are replaced with an amino acid different from the original peptide, one or two or more wild-type amino acids are deleted, one or two or more amino acids not present in the wild type are inserted, and / or amino acids not present in the wild type are added to the amino terminus (N terminus) and / or carboxyl terminus (C terminus) of the wild type (hereinafter collectively referred to as "mutations"). In the present application, "insertion" may also be included in "addition".

[0186] The term "CDR" (complementarity determining region), also known as "hypervariable region", refers to each region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. A natural four-chain antibody typically comprises six CDRs, three in the heavy chain variable region and three in the light chain variable region.

[0187] The term "variable region" refers to the variable region of an antibody, which is composed of two pairs of polypeptide chains, each pair having a heavy chain and a light chain. The N-terminal domain of each chain defines a region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition.

[0188] The term "Fab" refers to a constant domain containing the light chain (CL) and the first constant domain (CH1) of the heavy chain together with the variable domains VL (light chain variable region) and VH (heavy chain variable region) on the light chain and heavy chain, respectively. The variable domains contain the complementarity determining regions (CDRs) involved in antigen binding.

[0189] The term "scFv" includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the scFv further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0190] The term "ECD" is an extracellular domain. HER receptors are receptor protein tyrosine kinases belonging to the human epidermal growth factor receptor (HER) family and include EGFR, HER2, HER3 and HER4 receptors. HER2 receptors generally contain an extracellular domain that can bind to HER ligands, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxyl-terminal signaling domain with several tyrosine residues that can be phosphorylated. The extracellular domain of HER2 includes four domains, namely ECD1, ECD2, ECD3 and ECD4.

[0191] The term "antibody portion" refers to the antibody portion in the antibody drug conjugate, which, in certain specific embodiments, is connected to the intermediate linker portion through a specific functional group, and the antibody portion can specifically bind to the antigen.

[0192] The term "linker portion" refers to the portion of the antibody-drug conjugate that connects the antibody portion to the cytotoxic drug portion, and may be cleavable or non-cleavable. A cleavable linker refers to a linker that can be broken in the target cell to release the cytotoxic drug.

[0193] The term "cytotoxic drug moiety" refers to the cytotoxic drug moiety in the antibody-drug conjugate. In certain specific schemes, it is connected to the intermediate linker moiety through a functional group. In tumor cells, cytotoxic drug molecules will be released, thereby exerting an anti-tumor effect.

[0194] The term "trastuzumab": Trastuzumab, also known as trastuzumab, is a recombinant humanized monoclonal antibody that selectively targets the extracellular site of human epidermal growth factor receptor-4 (HER4) and can be used to treat HER2-positive cancers. An example of trastuzumab is sold under the trade name Marketed therapeutic monoclonal antibody products.

[0195] The term "Pertuzumab": The generic name Pertuzumab is a recombinant humanized monoclonal antibody that selectively acts on the extracellular site of human epidermal growth factor receptor-2 (HER2) and can be used to treat HER2-positive cancers.

[0196] The term "HER2" is the second member of the EGFR family and has tyrosine kinase activity. The HER2 expression level can be detected by immunohistochemistry. HER2 positive refers to IHC3+, and HER2 negative refers to IHC1+ / 0. For IHC2+, ISH testing should be performed for further clarification.

[0197] The term "cancer": refers to the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0198] The term "triple-negative breast cancer" refers to breast cancer that is negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2.

[0199] As used herein, unless otherwise specified, the terms "comprise, comprises and comprising" or their equivalents (contain, contains, containing, include, includes, including) are open-ended expressions, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0200] In this article, unless otherwise indicated, all numbers used herein expressing the amount of ingredients, measured values ​​or reaction conditions should be understood to be modified in all cases by the term "about". When connected with a percentage, the term "about" can mean, for example, ± 0.1%, preferably ± 0.05%, more preferably ± 0.01%.

[0201] Unless the context clearly indicates otherwise, singular terms include plural referents and vice versa. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0202] In the present context, the percent identity (degree of homology) between sequences can be determined by comparing two sequences, for example, using freely available computer programs commonly used for this purpose on the World Wide Web (eg www.ncbi.nlm.nih.gov), such as BLASTp or BLASTn with default settings. DETAILED DESCRIPTION

[0203] For the sake of clarity, the present application is further described with examples, but the examples do not limit the scope of the present application. The reagents used in the present application are generally commercially available and can be used without further purification.

[0204] The trastuzumab and pertuzumab used in the examples of this application are prepared according to conventional methods for antibodies, first constructing the vector, transfecting eukaryotic cells and then purifying and expressing. The sequence of trastuzumab is referenced to WHO DRUGINFORMATION INN RL78, and the sequence of pertuzumab is referenced to the examples section of WO0100245. DS-8201 is the active ingredient of Enhertu, a commercially available preparation of Daiichi Sankyo Co., Ltd., and its structure is the same as that of Trastuzumab-DXD prepared in this application (see Example 15 for the structure).

[0205] Example 1 Construction, expression and purification of anti-Her2 scFv-Fc and its variants

[0206] When constructing anti-Her2 scFv-Fc, the Fc part uses human IgG1, and the variable region sequence of the anti-Her2 arm is based on The sequence of the monoclonal antibody was converted into The light and heavy chain variable regions of the monoclonal antibody were connected in series to form anti-Her2 scFv-Fc (SEQ ID NO: 1), and its amino acid sequence is as follows:

[0207] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0208] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDI

[0209] QMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL

[0210] TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0211] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0212] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0213] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0214] In addition, point mutations were constructed on the anti-Her2 scFv-Fc sequence to construct the following variants:

[0215] anti-Her2-scFv-VL-F53Y-Fc (SEQ ID NO: 3): derived from wild-type anti-Her2 scFv-Fc, with F53Y mutation in the VL region; its amino acid sequence is as follows:

[0216] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0217] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDI

[0218] QMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTL

[0219] TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0220] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0221] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0222] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0223] anti-Her2-scFv-VL-F53A-Fc (SEQ ID NO: 5): derived from wild-type anti-Her2 scFv-Fc, with F53A mutation in the VL region; its amino acid sequence is as follows:

[0224] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0225] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDI

[0226] QMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASALYSGVPSRFSGSRSGTDFTL

[0227] TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0228] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0229] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0230] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0231] anti-Her2-scFv-VL-F53R-Fc (SEQ ID NO: 7): derived from wild-type anti-Her2 scFv-Fc, with F53R mutation in the VL region; its amino acid sequence is as follows:

[0232] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0233] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDI

[0234] QMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASRLYSGVPSRFSGSRSGTDFTL

[0235] TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0236] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0237] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0238] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0239] anti-Her2-scFv-VH-K30E-Fc (SEQ ID NO: 9): Derived from wild-type anti-Her2 scFv-Fc, with a K30E mutation in the VH region; its amino acid sequence is as follows:

[0240] EVQLVESGGGLVQPGGSLRLSCAASGFNIEDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0241] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDI

[0242] QMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL

[0243] TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

[0244] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[0245] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0246] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0247] The DNA sequences of anti-Her2 scFv-Fc and its variants (SEQ ID NOs: 2, 4, 6, 8 and 10) were synthesized and cloned into pcDNA3.1 expression vector respectively. TM The expression vector of anti-Her2 scFv-Fc or its variant was co-transfected into ExpiCHO cells (CHO-S, Thermo company) by expression kit (Thermo Fisher, catalog number A29133). The cells were cultured in an incubator with a humidified atmosphere of 8% CO2 at 37°C in ExpiCHO expression medium and on a tracked shaking platform rotating at 130 rpm. The culture supernatant was collected and protein A magnetic beads (Genscript, catalog number L00273) were used for protein purification. The protein concentration was measured by UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).

[0248] Table 1: Sequence information of anti-Her2 scFv-Fc and its variants

[0249]

[0250] Example 2 Aggregation verification of anti-Her2 scFv-Fc and its variants and binding detection with human Her2 antigen

[0251] The expressed and purified anti-Her2 scFv-Fc and its variants were tested for affinity to human Her2 protein using Biacore T200 (GE). The experimental process is described as follows:

[0252] A certain amount of anti-Her2 scFv-Fc or its variants were captured by a chip coupled with Anti-hIgG, and then the antigen human HER2 protein (Sino Biological, catalog number 10004-H08H) was passed through the chip surface, and the reaction signal was detected in real time by Biacore T200 to obtain the binding and dissociation curves. The buffer used in the experiment was Biacore universal buffer (137mM NaCl, 2.7mM KCl, 10mMNa2HPO4·12H2O, 1.8mM KH2PO4, 0.05% surfactant P20 (GE, catalog number BR-1000-54), pH 7.4). The reaction value of anti-hIgG (captured using a human antibody capture kit, GE, catalog number 29-2346-00) coupled to the CM5 chip surface reached about 9000RU, and the reaction value after capturing anti-Her2 scFv-Fc or its variants was about 200RU. Then the signal value of the interaction between different concentrations of human HER2 protein (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and anti-Her2 scFv-Fc or its variants was detected. The flow rate of the flow cell was 50μl / min, the binding time was 240s, the dissociation time was 1400s, and it was regenerated with 3M MgCl2 (GE Company) for 60s, and the baseline was stable. The results were obtained according to the affinity and kinetics 1:1 binding mode calculation in biacoreevaluation software. The affinity of anti-Her2scFv-Fc or variants with antigen human Her2 protein is shown in Table 2. Compared with anti-Her2 scFv-Fc (KD = 0.77nM), the F53A mutation has a greater effect on the binding affinity of the variant anti-Her2-scFv-VL-F53A-Fc (KD = 1.26nM) to the antigen human Her2 protein; the binding affinity of the variant anti-Her2-scFv-VL-F53Y-Fc (KD = 0.8nM) to the antigen human Her2 protein is similar to that of anti-Her2 scFv-Fc (KD = 0.77nM), and the KD of the variant anti-Her2-scFv-VH-K30E-Fc to the antigen human Her2 protein is 0.54nM. It can be seen that the introduction of F53Y and K30E mutations will not reduce the binding affinity to the antigen human Her2 protein.

[0253] The components of anti-Her2 scFv-Fc or its variants were separated by gel chromatography column, and the components were eluted in descending order of molecular weight. The column is ACQUITY UPLC Protein BEH SEC Column 1.7μm, 4.6*300mm gel chromatography column, column temperature 25℃. The mobile phase is 50mmol / L phosphate buffer-200mmol / L sodium chloride, pH 7.0 (weigh 2.33g of sodium dihydrogen phosphate dihydrate, 12.53g of disodium hydrogen phosphate dodecahydrate, 11.69g of sodium chloride, add about 800ml of ultrapure water, stir until fully dissolved, add ultrapure water to 1000ml, mix well and filter through a 0.22μm filter membrane). Dilute the sample to 10mg / ml with the mobile phase as the test solution, accurately measure 2μl and inject it into the liquid chromatograph (if the sample concentration is lower than 10mg / ml, adjust the injection volume to inject 20μg of protein), and detect at a wavelength of 280nm. The flow rate is 0.30ml / min, and the isocratic elution is 15min. For data processing, the results are quantitatively analyzed using the area normalization method. The peak area percentages of aggregates, immunoglobulin monomers and low molecular weight impurities were calculated respectively, where the aggregates were before the main peak, the immunoglobulin monomers were the main peaks, and the low molecular weight impurities were after the main peaks. The ratio of the main peak to the aggregate content of anti-Her2 scFv-Fc or its variants is shown in Table 2. The variants anti-Her2-scFv-VL-F53Y-Fc and anti-Her2-scFv-VH-K30E-Fc significantly reduced the aggregates, and the aggregates were reduced from 6.31% of anti-Her2 scFv-Fc to 4.94% and 3.39%, respectively.

[0254] Table 2: Aggregate content and binding affinity to antigen Her2 of anti-Her2 scFv-Fc and its variants

[0255]

[0256] Example 3 Construction, expression and purification of anti-Her2 bispecific antibodies

[0257] The anti-Her2 bispecific antibody was produced as human IgG1 by Knobs-into-holes (Ridgway, et al., 1996) Fc engineering. The Fc region sequence of one of the heavy chains was designed with H435R and Y436F mutations (Jendeberg et al., 1997) to reduce the affinity of Fc for protein A, which is beneficial for removing homodimers formed during the assembly process of the bispecific antibody in protein A affinity purification (patent US5945311A). It can be seen from Example 2 that the anti-Her2-scFv-VL-F53Y-Fc mutation and the anti-Her2-scFv-VH-K30E-Fc mutation can significantly reduce the anti-Her2-scFv aggregates without reducing the affinity for the Her2 antigen. In this embodiment, the antigen binding domain of one anti-Her2 arm of the anti-Her2 bispecific antibody is in the form of scFv (VH-linker-VL structure), and the variable region sequence contains the mutation K30E (b-anti-Her2-scFv-VH-K30E-Fc, SEQ ID NO: 21), the mutation F53Y (b-anti-Her2-scFv-VL-F53Y-Fc, SEQ ID NO: 23) or both of these point mutations anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO: 11). In this embodiment, the antigen binding domain of the other anti-Her2 arm of the anti-Her2 bispecific antibody is in the form of Fab, including anti-Her2-domain2-HC-Fc (SEQ ID NO: 13) and anti-Her2-domain2-LC (SEQ ID NO: 15); in addition, an anti-Her2 bispecific antibody was constructed using scFv (VH-linker-VL structure or VL-linker-VH structure) without mutation as a control.

[0258] Table 3: Sequence information of anti-Her2 bispecific antibodies

[0259]

[0260]

[0261]

[0262] The DNA sequences of anti-Her2 bispecific antibodies (SEQ ID NOs: 12, 14 and 16) were synthesized and cloned into the pcDNA3.1 expression vector. High-yield expression system (Cat. No.: MIR 6270) was used to co-transfect the expression vectors of anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO: 11), anti-Her2-domain2-HC-Fc (SEQ ID NO: 13) and anti-Her2-domain2-LC (SEQ ID NO: 15) into ExpiCHO cells at a transfection ratio of 1:1:1.5. The transfection density was 6×10 6 cells / ml. The culture medium is Expression medium (catalog number MIR 6200, manufacturer Mirus). Continue to culture until the 10th day after transfection and collect the cell culture supernatant by centrifugation. Protein purification was performed using protein A magnetic beads (Genscript, catalog number L00273). The protein concentration was measured by UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). This sample was named Expi Her2-2. Referring to this method, other bispecific antibodies Expi Her2-1, Expi Her2-3, Expi Her2-4, and Expi Her2-5 were expressed and purified.

[0263] Example 4 Preparation and verification of fucose knockout bispecific antibodies

[0264] Knocking out the fucose expression-related gene FUT8 can increase the interaction between IgG1 and FcgRIIIa, thereby enhancing the ADCC effect of the antibody (Shields et al., 2002; Yamane-Ohnuki et al., 2004). In this example, a fucose knockout anti-Her2 bispecific antibody was prepared using FUT8-knockout CHO-S cells (named CHO FUT8- / - cells). The DNA sequences of the anti-Her2 bispecific antibody (SEQ ID NOs: 12, 14 and 16) were synthesized and cloned into the pcDNA3.1 expression vector respectively. By using High-yield expression system (Cat. No.: MIR 6270) co-transfected the expression vectors of anti-Her2-scFv-VH-K30E-VL-F53Y-Fc, anti-Her2-domain2-HC-Fc and anti-Her2-domain2-LC into FUT8-knockout CHO-S cells at a transfection ratio of 1:1:1.5. The transfection density was 6×10 6 cells / ml. The culture medium is Expression medium (Cat. No. MIR 6200, manufacturer Mirus). Continue to culture until the 10th day after transfection and collect the cell culture supernatant by centrifugation. Protein purification was performed using protein A magnetic beads (Genscript, catalog number L00273). The protein concentration was measured by UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). This sample was named 23C2 Her2-2. Referring to this method, the sequences of Expi Her2-1, Expi Her2-3, Expi Her2-4, and Expi Her2-5 were expressed in CHOFUT8- / - cells, and the fucose knockout anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5 were obtained accordingly.

[0265] The anti-Her2 bispecific antibody samples expressed in CHOFUT8- / - cells and CHO-S cells were treated with the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, Mass., USA), the N-glycans were released from the protein, and then labeled. After separation by chromatographic column, the N-glycans were analyzed using an FLR detector (Waters, Milford, Mass., USA), and the structure and content of the N-glycans were obtained. The proportion of glycoform content is shown in Table 4. The proportion of fucose-free in the normal anti-Her2 bispecific antibody ExpiHER2-2 was 21.85%, and the proportion of fucose-free in the anti-Her2 bispecific antibody 23C2 HER2-2 expressed by CHO-S cells with FUT8- knocked out was 99.40%.

[0266] Table 4: Glycoform content analysis of anti-Her2 bispecific antibodies

[0267]

[0268] Example 5 Aggregation Verification of Bispecific Antibodies

[0269] This example relates to the aggregation validation of the anti-her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, and 23C2Her2-5.

[0270] The anti-her2 bispecific antibody was separated using a gel chromatography column to verify the aggregate content, and the components were eluted in descending order of molecular weight. The column is ACQUITY UPLC Protein BEH SEC Column 1.7μm, 4.6*300mm gel chromatography column, column temperature 25℃. The mobile phase is 50mmol / L phosphate buffer-200mmol / L sodium chloride, pH 7.0 (weigh 2.33g of sodium dihydrogen phosphate dihydrate, 12.53g of disodium hydrogen phosphate dodecahydrate, 11.69g of sodium chloride, add about 800ml of ultrapure water, stir until fully dissolved, add ultrapure water to 1000ml, mix well and filter through a 0.22μm filter membrane). Dilute the sample to 10mg / ml with the mobile phase as the test solution, accurately measure 2μl and inject it into the liquid chromatograph (if the sample concentration is lower than 10mg / ml, adjust the injection volume to inject 20μg of protein), and detect at a wavelength of 280nm. The flow rate is 0.30ml / min, and the isocratic elution is 15min. For data processing, the results are quantitatively analyzed using the area normalization method. The peak area percentages of aggregates, immunoglobulin monomers and low molecular weight impurities were calculated respectively, wherein the peak before the main peak was the aggregates, the main peak was the immunoglobulin monomers, and the peak after the main peak was the low molecular weight impurities.

[0271] Table 5: Aggregate content of anti-Her2 bispecific antibodies

[0272] Sample name Aggregates monomer Low molecular weight fragments 23C2 Her2-2 8.51％ 91.02％ 0.47％ 23C2 Her2-1 19.55％ 80.05％ 0.40％

[0273] The results showed that when scFV was assembled into a bispecific antibody, the resulting bispecific antibody still produced a large amount of aggregates, but the aggregate content of the bispecific antibody could be reduced by introducing mutations. Compared with 23C2Her2-1 without mutations, the aggregate content of 23C2 Her2-2 could be reduced to 8.51% (Table 5).

[0274] Example 6 Antigen Binding Detection of Anti-Her2 Bispecific Antibodies

[0275] The expressed and purified anti-Her2 bispecific antibody was used to measure the affinity of the test molecule to the HER2 protein by Biacore T200 (GE). The experimental process is described as follows:

[0276] A certain amount of anti-Her2 bispecific antibody was captured by a chip coupled with Anti-hIgG, and then human Her2 (SinoBiological, catalog number 10004-H08H) was passed over the chip surface, and the reaction signal was detected in real time using Biacore T200 to obtain the binding and dissociation curves. The buffer used in the experiment was Biacore universal buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4·12H2O, 1.8mM KH2PO4, 0.05% surfactant P20, pH 7.4). Anti-hIgG (captured using a human antibody capture kit, GE, catalog number 29-2346-00) was coupled to the CM5 chip surface with a reaction value of about 9000RU, and the reaction value after capturing the anti-Her2 bispecific antibody was about 200RU, and then the signal values ​​of the interaction between different concentrations of Her2 protein (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and the anti-Her2 bispecific antibody were detected. The flow rate of the flow cell was 50μl / min, the association time was 240s, the dissociation time was 1400s, and it was regenerated with 3M MgCl2 (GE) for 60s, and the baseline was stable.

[0277] The results were obtained by calculation using biacore evaluation software. The binding affinities of the anti-Her2 bispecific antibody 23C2Her2-2 and the control trastuzumab and pertuzumab to the antigen Her2 are shown in Table 6. The KD of the anti-Her2 bispecific antibody 23C2 Her2-2 to the antigen Her2 is 6.11E-10M, the KD of trastuzumab to the antigen Her2 is 1.22E-09M, and the KD of pertuzumab to the antigen Her2 is 2.35E-09M. The anti-Her2 bispecific antibody 23C2Her2-2 has a higher affinity for the antigen Her2 than trastuzumab and pertuzumab.

[0278] Table 6: Affinity of anti-Her2 bispecific antibodies for human Her2 antigen

[0279] ka(1 / Ms) kd(1 / s) KD(M) 23C2 Her2-2 1.79E+05 1.09E-04 6.11E-10 Trastuzumab control sample 1.73E+05 2.12E-04 1.22E-09 Pertuzumab control sample 1.15E+05 2.69E-04 2.35E-09

[0280] Example 7 Antigen Binding Detection of Anti-Her2 Bispecific Antibodies

[0281] Referring to the experimental process of Example 6, the affinity of the expressed and purified anti-Her2 bispecific antibodies 23C2Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5 to the HER2 protein can be determined by Biacore T200 (GE).

[0282] The results of affinity determination between the anti-Her2 bispecific antibody 23C2 Her2-1 and the antigen human Her2 protein are shown in Table 7. Combining the results of Tables 6 and 7, it can be seen that the introduction of F53Y and K30E mutations can improve the binding affinity of the anti-Her2 bispecific antibody to the antigen human Her2 protein.

[0283] Table 7: Affinity of anti-Her2 bispecific antibodies for human Her2 antigen

[0284] Sample name 23C2 Her2-1 Antigen Her2 KD 4.02nM

[0285] Example 8 Killing of Her2-positive target cells BT474 by anti-Her2 bispecific antibody

[0286] NK cells were provided by human PBMC (peripheral blood mononuclear cells) to study the killing effect of anti-Her2 bispecific antibodies on target cells (BT474Her2+++, source: Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences), and the in vitro activity of anti-Her2 bispecific antibodies was evaluated based on the EC50 size.

[0287] The specific experimental process is as follows: BT474 cells were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) to adjust the cell density to 3×10 5 cells / ml, 50 μl per well was inoculated in a 96-well cell culture plate (eppendorf, catalog number: 0030730199). Anti-Her2 bispecific antibodies of different concentrations (1000 ng / ml, 333 ng / ml, 111 ng / ml, 37 ng / ml, 12.3 ng / ml, 4.11 ng / ml, 1.37 ng / ml, 0.46 ng / ml, 0.15 ng / ml, 0.05 ng / ml) were prepared using 1640 experimental medium, and different concentrations of antibodies were added to the above 96-well cell culture plate, 50 μl per well. Human PBMCs were adjusted to a cell density of 1.5×10 using 1640 experimental medium. 6cells / ml, 100μl per well. Set up the drug administration group (target cell + effector cell + antibody), target cell group (BT474 cells), effector cell group (human PBMC), target cell + effector cell group, blank control group (culture medium) and lysate control group, target cell maximum release group (target cell + lysate), the effector-target ratio is 10:1. 45 minutes before the test, add 20μl / well lysate (Promega, catalog number G182A) to the target cell maximum release group and lysate control group. Use after 45 minutes. The cell lysis rate was detected by non-radioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780).

[0288] Lysis rate (%) = (OD 给药组 -OD 靶细胞+效应细胞组 ) / (OD 靶细胞最大释放组 -OD 靶细胞组 )×100%

[0289] Figure 1 The killing rate of anti-Her2 bispecific antibody on BT474 Her2+++ tumor cells is shown. ADCC-enhanced anti-Her2 bispecific antibody ( Figure 1 The killing effects of 23C2 HER2-1 and 23C2 HER2-2 on BT474 tumor cells were better than the combination of trastuzumab + pertuzumab and the anti-Her2 bispecific antibodies expressed in CHO-S (Expi HER2-1 and Expi HER2-2); the EC50 of the combination of trastuzumab + pertuzumab (1:1) was 8.627 ng / ml, the EC50 of Expi HER2-1 was 38.05 ng / ml, and Expi HER2-2 was better than Expi HER2-1, with an EC50 of 35.17 ng / ml; the EC50 of ADCC-enhanced 23C2 HER2-1 was 4.728 ng / ml, and the ADCC-enhanced 23C2 HER2-2 was better than the ADCC-enhanced 23C2 HER2-1, with an EC50 of 3.658 ng / ml.

[0290] Example 9 Killing of Her2-positive target cells NCI-N87 by anti-Her2 bispecific antibody

[0291] NK cells were provided by human PBMC (peripheral blood mononuclear cells) to study the killing effect of anti-Her2 bispecific antibodies on target cells (NCI-N87Her2++, source: Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences), and the in vitro activity of anti-Her2 bispecific antibodies was evaluated based on the EC50 size.

[0292] The specific experimental process is as follows: NCI-N87 cells were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) to adjust the cell density to 3×10 5 cells / ml, 50μl per well was inoculated in a 96-well cell culture plate (eppendorf, catalog number: 0030730199). Different concentrations (8.1nM, 2.7nM, 0.9nM, 0.3nM, 0.1nM, 0.03nM, 0.01nM, 0.003nM, 0.001nM, 0.0004nM) of anti-Her2 bispecific antibodies or control drugs were prepared using experimental culture medium, and different concentrations of antibodies or control drugs were added to the above 96-well cell culture plate, 50μl per well. Human PBMCs were adjusted to a cell density of 1.5×10 using experimental culture medium. 6 cells / ml, 100μl per well. Set up the drug administration group (target cell + effector cell + antibody or control drug), target cell group (NCI-N87 cells), effector cell group (human PBMC), target cell + effector cell group, blank control group (culture medium) and lysate control group, target cell maximum release group (target cell + lysate), the effector-target ratio is 10:1. 45 minutes before the test, add 20μl / well lysate (Promega, catalog number G182A) to the target cell maximum release group and lysate control group. Use after 45 minutes. The cell lysis rate was detected by non-radioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780). Trastuzumab, T-DM1 (trastuzumab-emtansine conjugate, trade name ), Trastuzumab + Pertuzumab (1:1), and Expi HER2-1 were used as control drugs.

[0293] Lysis rate (%) = (OD 给药组 -OD 靶细胞+效应细胞组 ) / (OD 靶细胞最大释放组 -OD 靶细胞组 )×100%

[0294] Figure 2The killing rate of anti-Her2 bispecific antibodies on NCI-N87 tumor cells is shown. The ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 is better than the combination of trastuzumab + pertuzumab, trastuzumab, T-DM1 and Expi HER2-1 in killing NCI-N87 tumor cells; among them, the EC50 of ADCC-enhanced 23C2 HER2-2 is 0.02447nM, the EC50 of trastuzumab + pertuzumab combination is 0.08267nM, the EC50 of Expi HER2-1 is 0.1048nM, the EC50 of T-DM1 is 0.07392nM, and the EC50 of trastuzumab is 0.07468nM.

[0295] Example 10 Anti-Her2 bispecific antibody kills trastuzumab-resistant cells JIMT-1

[0296] NK cells were provided by human PBMC (peripheral blood mononuclear cells) to study the killing effect of anti-Her2 bispecific antibodies on target cells (JIMT-1, source: AddexBio, Catalog #: C0006005), and the in vitro activity of anti-Her2 bispecific antibodies was evaluated based on the EC50 size.

[0297] The specific experimental process is as follows: JIMT-1 cells were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) to adjust the cell density to 3×10 5 cells / ml, 50μl per well was inoculated in a 96-well cell culture plate (eppendorf, catalog number: 0030730199). Different concentrations (8.1nM, 2.7nM, 0.9nM, 0.3nM, 0.1nM, 0.03nM, 0.01nM, 0.003nM, 0.001nM, 0.0004nM) of anti-Her2 bispecific antibodies or control drugs were prepared using experimental culture medium, and different concentrations of antibodies or control drugs were added to the above 96-well cell culture plate, 50μl per well. Human PBMCs were adjusted to a cell density of 1.5×10 using experimental culture medium. 6 cells / ml, 100μl per well. Set up the drug administration group (target cell + effector cell + antibody or control drug), target cell group (BT474 cells), effector cell group (human PBMC), target cell + effector cell group, blank control group (culture medium) and lysate control group, target cell maximum release group (target cell + lysate), the effector-target ratio is 20:1. 45 minutes before the test, add 20μl / well lysate (Promega, catalog number G182A) to the target cell maximum release group and lysate control group. Use after 45 minutes. The cell lysis rate was detected by non-radioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780). Trastuzumab, T-DM1, trastuzumab + pertuzumab (1:1), and ExpiHER2-1 were used as control drugs.

[0298] Lysis rate (%) = (OD 给药组 -OD 靶细胞+效应细胞组 ) / (OD 靶细胞最大释放组 -OD 靶细胞组 )×100%

[0299] Figure 3 The killing rate of anti-Her2 bispecific antibodies on JIMT-1 tumor cells was shown. The anti-Her2 bispecific antibody 23C2 HER2-2 with ADCC enhancement had better killing effect on JIMT-1 tumor cells than the combination of trastuzumab + pertuzumab, trastuzumab, T-DM1, and ExpiHER2-1; the EC50 of ADCC enhancement of 23C2 HER2-2 was 0.01006nM, the EC50 of the combination of trastuzumab + pertuzumab was 0.06727nM, the EC50 of Expi HER2-1 was 0.08066nM, the EC50 of T-DM1 was 0.08357nM, and the EC50 of trastuzumab was 0.07443nM. In addition, the anti-Her2 bispecific antibody 23C2 HER2-2 had a higher cell lysis rate.

[0300] Example 11 Inhibition of proliferation of BT474 Her2+++ tumor cells by anti-Her2 bispecific antibody

[0301] 23C2 Her2-2, trastuzumab, and pertuzumab were diluted to a final concentration of 3.2 μg / ml using DMEM / F12 medium (GIBCO, catalog number 11330-032) containing 2% FBS (fetal bovine serum, manufacturer GIBCO, catalog number: 10099-141), and then diluted to 9 concentrations (1.6 μg / ml, 0.8 μg / ml, 0.4 μg / ml, 0.2 μg / ml, 0.1 μg / ml, 0.05 μg / ml, 0.025 μg / ml, 0.0125 μg / ml, 0.00625 μg / ml) in a 1:1 ratio. BT474 Her2+++ cells in the logarithmic growth phase were taken and the density was adjusted to 1×10 5cells / ml for plating, add 100μl to each well, and set up a blank well without cells as a control. Add the above gradient diluted samples, 50μl to each well. Culture in an incubator at 37°C and 5% CO2 for 5 days. Discard the culture medium, add CCK-8 (Japan Tongren Chemical, product number: CK04) working solution, 100μl to each well, incubate for 4 to 5 hours and then place in an ELISA reader (manufacturer Thermo, model: VarioskanFlash), use 630nm as the reference wavelength, read and record the absorbance value of the well plate at a wavelength of 450nm. Calculate the tumor cell proliferation inhibition rate.

[0302] Results Figure 4 The ADCC-enhanced anti-Her2 bispecific antibody 23C2 Her2-2 had a proliferation inhibition rate of 78.38% on BT474 tumor cells, which was superior to the proliferation inhibition rate of trastuzumab (54.12%) and the proliferation inhibition rate of trastuzumab + pertuzumab combined with trastuzumab (53.7%).

[0303] Example 12 Inhibitory effect of anti-Her2 bispecific antibody on NCI-N87 Her2++ gastric cancer xenografts in nude mice

[0304] The in vivo efficacy of anti-Her2 bispecific antibodies was evaluated in a mouse xenograft model using NCI-N87 Her2++ gastric cancer cells (cell bank of the Chinese Academy of Sciences). NCI-N87 Her2++ gastric cancer cells were prepared at a concentration of 5×10 7 pcs / ml*0.1ml / mouse, inoculated into the right armpit of nude mice (from Changzhou Cavens Experimental Animal Co., Ltd., nude mice, 14-17g, male, raised in SPF grade) under sterile conditions. The diameter of the transplanted tumor of nude mice was measured with a vernier caliper. When the tumor grew to 100-250mm 3 The animals were then divided into 5 groups:

[0305] Group 1: Control

[0306] Group 2: Expi Her2-1, 10 mg / kg

[0307] Group 3: 23C2 Her2-2, 5 mg / kg

[0308] Group 4: 23C2 Her2-2, 10 mg / kg

[0309] Group 5: Per+Tra (trastuzumab + pertuzumab), 5 mg / kg + 5 mg / kg

[0310] Each group was intravenously administered at the corresponding dose, twice a week, for about 3 weeks (6 times). Except for Group 5, the two drugs were administered at a volume of 5 ml / kg, and the other groups were administered at a volume of 10 ml / kg. Group 1 was also given 10 ml / kg PBS (Hyclone; Catalog No.: sh30256.01) by IV. When administering the combination group, trastuzumab was administered at least 30 minutes after pertuzumab.

[0311] The tumor diameter measurement method was used to dynamically observe the anti-tumor effect of the test substance. The tumor volume was measured 2-3 times a week, and the mice were weighed and the data were recorded. The general performance of the mice was observed daily.

[0312] Detection indicators:

[0313] Tumor volume (TV), calculated as follows: TV = 1 / 2 × a × b 2 , where a and b represent the length and width of the tumor, respectively.

[0314] In this experiment, the drug was administered from day 0, and a total of 6 times (days 0, d3, d7, d10, d14, and d17) were administered. No animal died until day 21 of the experiment. The average weight of mice in each group showed an increasing trend ( Figure 6 ). The drug had no obvious toxic effects.

[0315] The effects of the test samples of group 2 (10 mg / kg), group 3 (5 mg / kg), group 4 (10 mg / kg), and Per+Tra combination group (5 mg / kg+5 mg / kg) on ​​the volume of NCI-N87 gastric cancer nude mice xenografts are shown in Table 8 and Table 9. Figure 5 The 23C2Her2-2 group (10 mg / kg) was superior to the Expi Her2-1 (10 mg / kg) and Per+Tra combination group (5 mg / kg+5 mg / kg) in inhibiting NCI-N87 gastric cancer xenograft tumors in nude mice.

[0316] Table 8: Effects of anti-HER2 dual antibodies on the volume of NCI-N87 gastric cancer xenografts in nude mice (mean±SD)

[0317]

[0318] Example 13 Synthesis of MC-GGFG-DXD

[0319]

[0320] Step 1 A5 (Synthesis of ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl acetate)

[0321] Weigh 20g SM5 (N-[(9H-fluorene-9-ylmethoxy)carbonyl]glycylglycine) into a 1L three-necked flask, add 300ml tetrahydrofuran and 100ml toluene, stir evenly, add 30g lead tetraacetate and 5.4g pyridine. Heat to 65℃ and react for 4 hours. Cool to room temperature and filter out the solid. Concentrate the organic phase to dryness at 40℃. Add 300ml ethyl acetate and 300ml water to the concentrated organic phase. Stir for 20 minutes. Separate the ethyl acetate phase. Add 100ml saturated sodium chloride solution to the ethyl acetate phase and stir for 20 minutes. Separate the ethyl acetate phase and concentrate. Perform silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 13g A5. Yield 63%. ESI-MS: m / z = 391.1 [M+Na] + .

[0322] Step 2: Synthesis of B5 ([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]benzyl acetate)

[0323] Weigh 1.0g A5 and add it to a 250ml single-necked flask, add 15ml DME (ethylene glycol dimethyl ether), add 0.897g benzyl glycolate, put it in an ice-water bath and cool it to 0℃. Prepare a solution with 0.27ml water and 0.108g NaOH. Add the prepared NaOH solution to the reaction solution. React at 0℃ for 1 hour, add 0.078g glacial acetic acid to the reaction solution. Add 100ml water and 100ml ethyl acetate, stir at room temperature for 20 minutes, separate the organic phase and concentrate it to dryness. Silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 0.68g B5. Yield 53%. ESI-MS: m / z = 497.1 [M+Na] + .

[0324] Step 3 Synthesis of C5 ([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid)

[0325] Weigh 0.68g of B5 and add it to a 250ml hydrogenation bottle, add 20ml of ethanol and 10ml of ethyl acetate, and add 0.34g of wet palladium carbon (palladium content 10%). Use a hydrogen balloon to pass hydrogen. React at room temperature for 1 hour. Filter off the palladium carbon with diatomaceous earth. Concentrate the filtrate to dryness at 40°C. Obtain 425mg of C5. Yield 77%. ESI-MS: m / z=407.1[M+Na] + .

[0326] Step 4 Synthesis of D5 ([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6'7']indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycine amide)

[0327] Weigh 50 mg SM4 (isatecan mesylate) and 50 mg C5 into a 100 ml single-necked flask, add 2 ml DMF (N, N-dimethylformamide), cool to 0°C, add 54 mg HATU (2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate) and 30 mg DIEA (N, N-diisopropylethylamine). Return to room temperature. React at room temperature for 3 hours. Add 100 ml dichloromethane and 100 ml water. Stir for 20 minutes. Separate the organic phase and concentrate to dryness. Silica gel column chromatography (dichloromethane: methanol = 10: 1) to obtain 88 mg D5. Yield 84%. ESI-MS: m / z = 802.4 [M+H] + .

[0328] Step 5 Synthesis of E5 ({[(glycyl)amino]methoxy}acetyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6'7']indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide)

[0329] Weigh 78 mg of D5 into a 100 ml single-necked flask, add 4 ml of tetrahydrofuran, cool to 0°C, add 78 mg of ethylenediamine. Return to room temperature. React for 5 hours. Concentrate the reaction solution to dryness at 40°C. Obtain 55 mg of E5. Yield 98%. ESI-MS: m / z=580.3[M+H] + Step 6 Synthesis of MC-GGFG-DXD

[0330] Weigh 55 mg of E5 and add it to a 100 mg single-necked flask, add 2 ml of N,N-dimethylformamide, add 48 mg of MC-GGF-OH (maleimidocaproyl glycyl glycyl phenylalanine), and cool to 0°C. Add 54 mg of HATU and 30 mg of DIEA. Return to room temperature. React for 1 hour. Add 100 ml of ethyl acetate and 100 ml of water and stir for 20 minutes. Separate the organic phase. Concentrate it to dryness at 40°C. Silica gel column chromatography (ethyl acetate: methanol = 10:1) gave 26 mg of MC-GGFG-DXD. Yield 27%. ESI-MS: m / z = 1034.51 ([M+H] + ). 1 H-NMR(500MHz,DMSO-d6)8.62(1H,t,J=6.5Hz),8.50(1H,d,J=9.0Hz),8.29 (1H,t,J=6.0Hz),8.12(1H,d,J=8.0Hz),8.06(1H,t,J=6.0Hz),8.00(1H,t,J =6.0Hz),7.76(1H,d,J=11Hz),7.30(1H,s),7.25~7.15(5H,m),6.90(2H,s), 6.52(1H,brs),5.61~5.57(1H,m),5.42~5.40(2H,m),5.19~5.16(2H,m),4.6 4(2H,d,J=7.0Hz),4.49~4.44(1H,m),4.05~4.01(2H,m),3.76~3.51(6H,m) ,3.37~3.32(2H,m),3.21~3.11(2H,m),3.02(1H,dd,J=4.5,14.0),2.77(1H, dd,J=9.5,13.5),2.37(3H,s),2.21~2.15(2H,m),2.09(2H,t,J=7.5Hz),1.9 1~1.81(2H,m), 1.49~1.42(4H,m), 1.20~1.14(2H,m), 0.87(3H,t,J=6.5Hz).

[0331] Example 14 Synthesis of deuterated MC-GGFG-DXD (MC-GGFG-DDDXD), deuterated DXD (DDDXD) and DXD

[0332]

[0333] Step 1 Synthesis of Intermediate A

[0334] Under nitrogen protection, add 80g of ethyl diazoacetate to a 3L single-mouth bottle, add 800ml of dichloromethane and 800ml of 1% deuterated acetic acid solution (8g of deuterated acetic acid dissolved in 800ml of deuterated water). Stir the reaction solution at room temperature under light-shielding conditions for 75 hours. Separate and collect the organic phase, extract the aqueous phase with dichloromethane twice (200ml*2), and combine the organic phases. Wash the organic phase with 200ml of deuterated water, dry the obtained organic phase with anhydrous sodium sulfate, filter to remove sodium sulfate, and concentrate the filtrate to dryness under reduced pressure at 20℃ to obtain 49.25g of intermediate A. The yield is 66%. 1 H NMR (500 MHz, CDCl3) δ4.27 (q, J = 7.1 Hz, 1H), 1.31 (t, J = 7.1 Hz, 2H).

[0335] Step 2 Synthesis of Intermediate B

[0336] Weigh 50g of N-fluorenylmethoxycarbonyl-glycyl-glycine and add it to a 2L round-bottom flask, add 750ml of tetrahydrofuran and 150ml of glacial acetic acid, stir at 40°C for 20 minutes, add 100g of lead tetraacetate, heat to 80°C and continue to react for 3 hours. Cool to room temperature, filter with suction, and wash the filter cake with 250ml of ethyl acetate. Concentrate the filtrate to dryness. Add 330ml of dichloromethane and 670ml of ethyl acetate to dissolve and obtain the organic phase. Wash the organic phase with 30% potassium bicarbonate aqueous solution 3 times (500ml*3). Dry the organic phase with anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure, add 100ml of dichloromethane to dissolve, then add 100ml of n-hexane, stir at room temperature until solid precipitates, then add 300ml of a mixed solution of n-hexane and dichloromethane (n-hexane: dichloromethane = 1:1), and stir overnight. Filter and dry the filter cake in a vacuum oven at 40°C for 4 hours to obtain 37.3 g of intermediate B. Yield: 72%. LCMS (ESI) m / z: 391.09 [M+Na] + .

[0337] Step 3 Synthesis of Intermediate C

[0338] Weigh 78g of intermediate B into a 3000ml single-necked flask, add 800ml of dichloromethane, and add 45g of compound A. Put the 3000ml single-necked flask into an ice-water bath and cool it to 0°C. Dissolve 16g of lithium tert-butoxide in 400ml of dichloromethane to prepare a lithium tert-butoxide solution. Add the lithium tert-butoxide solution to a 3000ml round-bottom flask. React at 0°C for 3 hours. Move to room temperature, add 800ml of water and stir. Separate the liquid and collect the organic phase. The aqueous phase is extracted with 400ml of dichloromethane. Combine the organic phases. The organic phase is washed once with 800ml of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. Silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) gives 61g of intermediate C. Yield 70%. LCMS (ESI) m / z: 437.34 [M+Na] + .

[0339] Step 4 Synthesis of Compound D

[0340] 31.2 g of compound C was added to 270 mL of deuterated methanol and 70 mL of heavy water, stirred under ice bath, then 5.5 g of NaOH was added, and stirred at room temperature overnight. 300 mL of ethyl acetate and 300 mL of water were added to the reaction solution for extraction, and 20 mL of glacial acetic acid was added to the aqueous layer to adjust the pH to 2-3. Solids precipitated and were filtered to obtain 20.3 g of compound D. Yield 69.8%. LCMS (ESI) m / z: 409.08 [M+Na] + .

[0341] Step 5 Synthesis of Compound E

[0342] Weigh 2.0g of exatecan mesylate dihydrate and 1.63g of compound D and add them to a 100ml round-bottom flask. Add 40ml of N,N-dimethylformamide and stir. Cool to 0℃. Add 2.0g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 1.82g of N,N-diisopropylethylamine in sequence. React at 0℃ for 3h. Pour the reaction solution into 120ml of ice water. Stir for 1h. Filter. Dissolve the filter cake with dichloromethane. Column chromatography (100g of 100-200 mesh silica gel, dichloromethane: methanol = 30:1, 2L). Obtain 2.6g of compound E. Yield 92%. LCMS (ESI) m / z: 804.84 [M+H] + .

[0343] Step 6 Preparation of Compound F

[0344] Weigh 0.38g of 1,8-diazabicyclo[5.4.0]undec-7-ene and add it to a 100ml round-bottom flask. Add 20ml of tetrahydrofuran to the round-bottom flask and stir. Cool to 0°C. Weigh 2.0g of compound E and prepare a solution with 20ml of tetrahydrofuran. Slowly add the prepared solution of compound E to a 100ml round-bottom flask. Naturally warm the reaction solution to room temperature and react for 3h. Filter under nitrogen protection. Obtain 1.45g of compound F. Yield 98%. LCMS (ESI) m / z: 582.39 [M+H] + .

[0345] Step 7 Preparation of Compound H

[0346] Weigh 1.00g of compound F and 0.97g of compound G into a 100ml round-bottom flask, and add 10ml of N,N-dimethylformamide. Cool to -20℃, add 0.34g of 1-hydroxybenzotriazole and 0.49g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. React at -20℃ for 3h. Add 20ml of DCM and 20ml of water to the reaction solution, stir for 0.5h, let stand, separate the liquids, and collect the organic phase. Dry the organic phase with anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure. Silica gel column chromatography (dichloromethane: methanol = 15:1) to obtain 400mg of compound H. The yield is about 22%. MS m / s: 1037.08 [M+H] + . H-NMR (500MHz, DMSO-d6)8.62(1H,t,J=6.5),8.49(1H,d,J=8.5),8.29(1H,t,J=5.5),8.12(1H,d,J=8.0),8.06(1H,t,J=5.5),8.00(1H,t,J= 5.5),7.74(1H,d,J=10.5),7.30(1H,s),7.27~7.12(5H,m),6.98(2H,s ),6.51(1H,brs),5.61~5.58(1H,m),5.45~5.37(2H,m),5.22~5.13(2H, m), 4.64 (2H, d, J = 6.5), 4.49~4.45 (1H, m), 3.76~3.57 (6H, m), 3.37~3. 32(2H,m),3.24~3.09(2H,m),3.02(1H,dd,J=4.5,14.0),2.77(1H,dd,J =9.5,13.5),2.36(3H,s),2.23~2.14(2H,m)2.09(2H,t,J=7.5),1.91~ 1.79(2H,m), 1.49~1.42(4H,m), 1.20~1.14(2H,m), 0.87(3H,t,J=7.5).

[0347]

[0348]

[0349] Step 1 Synthesis of Intermediate I

[0350] 0.85 g of compound A was added to 9 ml of deuterated methanol and 1 ml of heavy water, stirred under ice bath, then 0.32 g of sodium hydroxide was added, stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure at 40°C to obtain compound I, which was directly used for the next step without purification.

[0351] Step 2 Synthesis of compound J(DDDXD)

[0352] 0.30 g of exatecan mesylate dihydrate and 0.056 g of intermediate I were added to 3 ml of N,N-dimethylformamide. Stirred under ice bath, then added 0.32 g of 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate and 0.22 g of N,N-diisopropylethylamine, and stirred at room temperature for 4 h. The reaction solution was prepared by liquid phase to obtain 0.18 g of compound J. The yield was 64.3%. LC-MS (ESI) m / z: 496.37 [M+H] + . 1 H-NMR (500MHz, DMSO-d6)8.39(d,J=8.9Hz,1H),7.70(d,J=10.9Hz,1H),7.29(s,1H),6.52(s,1H),5.58~5.54(m,1H),5.47(s,1H) ), 5.40 (s, 2H), 5.17 ~ 5.02 (m, 2H), 3.24 ~ 3.03 (m, 2H), 2.34 (s, 3H), 2.25 ~ 2.09 (m, 2H), 1.92 ~ 1.80 (m, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0353] In addition, DXD was prepared according to the method disclosed in Example 76 in the specification of patent WO2014057687.

[0354]

[0355] Example 15 Preparation of Antibody Drug Conjugates

[0356] Reagents:

[0357] Solution A: pH 7.4 PBS buffer

[0358] Solution B: 10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution

[0359] Solution C: DMSO (dimethyl sulfoxide)

[0360] Solution D: Histidine buffer (containing 0.89 mg / ml L-histidine and 4.04 mg / ml L-histidine hydrochloride monohydrate)

[0361] Solution E: 700 mg / ml sucrose solution (prepared with solution D)

[0362] Solution F: 20 mg / ml Tween 80 (prepared with solution D)

[0363] Antibodies: Trastuzumab, 23C2 Her2-2

[0364] Linker-payload (linker-cytotoxic drug part): MC-GGFG-DXD, MC-GGFG-DDDXD

[0365] Table 9. Experimental conditions and groups:

[0366]

[0367] Experimental process:

[0368] 1. Antibody replacement

[0369] a. Fully wet the 30KD ultrafiltration centrifuge tube with solution A;

[0370] b. Replace the antibody into solution A;

[0371] c. Add appropriate amount of solution A to adjust the antibody concentration to 5 mg / ml (23C2 Her2-2) and 7.5 mg / ml (trastuzumab).

[0372] 2. Antibody reduction

[0373] a. Calculate the molar amount of antibody, recorded as N1;

[0374] b. Add an appropriate amount of solution B to the antibody solution so that the TCEP molar amount of the reaction system is N2;

[0375] c. Wrap with aluminum foil, place on a rotary incubator and shake at low speed (20 rpm) at 37°C in the dark for 1 hour.

[0376] 3. Coupling

[0377] a. Take an appropriate amount of linker-payload and dissolve it in DMSO to a final concentration of 10 mg / ml;

[0378] b. Add DMSO to the antibody solution to make the antibody concentration 5wt%, and then add an appropriate amount of linker-payload solution to make the molar concentration

[0379] N3;

[0380] c. Wrap with aluminum foil, place on a rotary incubator and shake at low speed (20 rpm) at 20°C in the dark for 2 h.

[0381] 4. Coupling termination

[0382] a. Wet the ultrafiltration centrifuge tube with solution D;

[0383] b. Replace the antibody into solution D, add appropriate amount of solution E and F and adjust the concentration of sucrose and Tween 80 to 90 mg / ml and 90 mg / ml respectively.

[0384] 0.3mg / ml, stored at -80℃.

[0385] Determination of DAR value (average number of drug connections per antibody molecule) of antibody-drug conjugates

[0386] The DAR value was determined by LC-MS method. 50 μg of the prepared ADC sample was taken, 1 μl of glycosidase PNGase F (Ruian Bio, China) was added, and incubated at 37°C for 20 hours. The mass spectrometer in the experiment was a high-resolution Xevo G2-XS (Waters, USA). The sample concentration was adjusted to 5 μM, and the direct injection method was used to collect mass spectrometry data in positive ion mode. The collected non-denaturing mass spectrometry data was analyzed and processed using the software UNIFI1.8.2.169 (Waters, USA).

[0387] Protein concentration determination of antibody drug conjugates

[0388] The protein concentration was detected by the Lowry method. Trastuzumab and 23C2 Her2-2 were used as standard products. The absorbance values ​​of the standard products and the prepared ADC samples at OD650 wavelength were measured by an ELISA instrument, and the standard curve was fitted. The sample absorbance values ​​were substituted into the standard curve to calculate the protein concentration.

[0389] The following antibody drug conjugates were prepared and tested by the above method:

[0390]

[0391] Trastuzumab-DXD, antibody concentration: 4.25 mg / ml, DAR: 7.6.

[0392]

[0393] Trastuzumab-DDDXD, antibody concentration: 4.29 mg / ml, DAR: 7.7.

[0394]

[0395] 23C2 Her2-2-DXD, antibody concentration: 4.35 mg / ml, DAR: 5.7.

[0396]

[0397] 23C2 Her2-2-DDDXD, antibody concentration: 4.16mgml, DAR: 5.8.

[0398] Example 16 In vitro enzyme activity of deuterated DXD (DDDXD)

[0399] 1. Preparation of reagents and materials

[0400] a. Prepare 1% agarose electrophoresis gel;

[0401] b. Prepare gelred dyeing solution and store it away from light;

[0402] c. Preparation of topoisomerase I working solution: prepare with ultrapure water and buffer.

[0403] 2. Sample preparation

[0404] a. Redissolve and dilute the compound (DDDXD) with DMSO. The compound concentration starts from 200 μM, and is diluted 10 times with 5 gradients.

[0405] 3. Reaction system

[0406] a. Positive control: 15 μl ultrapure water + 2 μl 10× DNA Topoismerasel Buffer + 2 μl 0.1% BSA + 1 μl pBR322 DNA;

[0407] b. Negative control: 14 μl ultrapure water + 2 μl 10× DNA Topoisomerase Buffer + 2 μl 0.1% BSA + 1 μl pBR322 DNA + 1 μl topoisomerase I working solution;

[0408] c. Sample group: 12 μl ultrapure water + 2 μl 10× DNA Topoismerasel Buffer + 2 μl 0.1% BSA + 1 μl pBR322 DNA + 1 μl topoisomerase I working solution + 2 μl compound.

[0409] 4. Experimental process

[0410] a. Place the above reaction system in a 37°C water bath for 30 min;

[0411] b. Add 2 μl loading buffer to each tube to terminate the reaction;

[0412] c. Agarose gel electrophoresis at a voltage of 2-2.5 V / cm for 1.5 h;

[0413] d. Stain the gel after electrophoresis with GelRed in the dark for 1.5 h and take pictures with a gel imager.

[0414] Example 17 Antigen Binding Detection of Antibody Drug Conjugates

[0415] Referring to the method of Example 6, the affinities of Trastuzumab, 23C2 HER2-2, Trastuzumab-DDDXD and 23C2HER2-2-DDDXD to HER2 protein were measured and are shown in Table 10 below:

[0416] sample ka(1 / Ms) kd(1 / s) KD(M) Trastuzumab 1.23E+05 1.12E-04 9.16E-10 23C2 HER2-2 1.73E+05 6.06E-05 3.49E-10 Trastuzumab-DDDXD 1.28E+05 1.19E-04 9.25E-10 23C2 HER2-2-DDDXD 1.28E+05 3.26E-05 2.55E-10

[0417] The results showed that the anti-Her2 bispecific antibody had stronger antigen-binding activity than trastuzumab, and the anti-Her2 bispecific antibody ADC had stronger antigen-binding activity than trastuzumab ADC.

[0418] Example 18 Endocytosis experiment of antibody drug conjugate

[0419] Experimental method: Take one bottle of cells in the logarithmic growth phase (NCI-N87 cells and SK-BR-3 cells) and adjust the cell density to 2.5×10 6 cells / ml, 20μl / well was added to a 96-well plate. The ADC samples Trastuzumab-DDDXD and 23C2 HER2-2-DDDXD prepared in Example 15 were pre-diluted to a concentration of 40μg / ml, labeled as S1, and then 3-fold gradient dilution was performed to obtain the corresponding samples S1-S9. In addition, DS-8201 and control IgG1 (a non-HER2 target-specific IgG1; Yiqiao Shenzhou, catalog number HG1K) and DDDXD conjugate IgG1-DDDXD were used as controls. The gradient diluted sample solution and the labeled endocytosis reagent (sartorius, 90564) were added to the cell plate, 20μl was added to each well, and incubated at 37°C for 15min. Take out the 96-well cell culture plate, add the above two cells at 20μl / well, place the cell culture plate at 37°C, and continue to incubate for 2h. Take out the 96-well cell culture plate, place it in a flow cytometer, and measure the signal intensity.

[0420] The results of the endocytosis experiment on two HER2-positive cells, NCI-N87 and SK-BR-3, are shown in Figure 8 and Fig. 9 The results showed that the internalization of anti-Her2 dual antibody ADC was stronger than that of trastuzumab ADC.

[0421] Example 19 Deuterated DXD and Cellular Activity of Antibody Drug Conjugates

[0422] DXD and DDDXD were pre-diluted to 140,000 ng / ml using culture medium, labeled as S1, and then five-fold gradient dilution was performed to obtain the corresponding reference samples S1-S9. The final drug concentration range was 35,000 ng / ml to 0.0896 ng / ml, with a total of 9 concentrations. HER2-positive tumor cells NCI-N87 in the logarithmic growth phase were taken and the density was adjusted to 1×10 5 cells / ml for plating, add 100μl to each well, and set up a blank well without cells as a control. Add the above two samples in gradient dilution, 50μl to each well. Culture in a carbon dioxide incubator at 37°C and 5% CO2 for 5 days. Discard the culture medium, add CCK-8 (Japan Tongren Chemical, product number: CK04) working solution, 100μl to each well, incubate for 4 to 5 hours and then place in an ELISA reader (manufacturer Thermo, model: VarioskanFlash), use 630nm as the reference wavelength, read and record the absorbance value of the well plate at a wavelength of 450nm. Calculate the tumor cell proliferation inhibition rate.

[0423] The results of the NCI-N87 cell experiment are shown in Table 11 below:

[0424] sample <![CDATA[IC 50 (nM)]]> DXD 4.45 DDXD 4.01

[0425] DDDXD showed stronger tumor cell proliferation inhibitory activity than DXD.

[0426] The antibody to be tested and the antibody drug conjugate prepared in Example 15 were pre-diluted to 20 μg / ml using culture medium, marked as S1, and then five-fold gradient dilution was performed to obtain the corresponding samples S1 to S9. The final drug concentration range was 5000 ng / ml to 0.0128 ng / ml, with a total of 9 concentrations. HER2-positive tumor cells (NCI-N87, BT474 and SK-BR-3) in the logarithmic growth phase were taken and the density was adjusted to 2×10 4 cells / ml for plating, add 100μl to each well, and set up a blank well without cells as a control. Add gradient diluted samples, 50μl to each well. Culture in a carbon dioxide incubator at 37°C and 5% CO2. Discard the culture medium, add CTG detection solution (Promega, catalog number: G7572), 100μl to each well, incubate for 10 minutes, place on a multi-function plate reader (manufacturer Thermo, model: VarioskanFlash), and read the chemiluminescence value. Calculate the tumor cell proliferation inhibition rate.

[0427] The results of the NCI-N87 cell experiment are shown in Table 12 below:

[0428] sample Inhibition rate % Trastuzumab 34.51 Trastuzumab-DDDXD 70.81 23C2 HER2-2 75.73 23C2 HER2-2-DDDXD 90.08

[0429] The results of the BT474 cell experiment are shown in Table 13 below:

[0430] sample Inhibition rate % Trastuzumab 67.91 Trastuzumab-DDDXD 47.82 23C2 HER2-2 75.56 23C2 HER2-2-DDDXD 73.76

[0431] The results of the SK-BR-3 cell experiment are shown in Table 14 below:

[0432] sample Inhibition rate % Trastuzumab 20.36 Trastuzumab-DDDXD 67.15 23C2 HER2-2 62.89 23C2 HER2-2-DDDXD 75.56

[0433] From the above results, it can be seen that the cell killing ability of anti-Her2 dual antibody ADC is better than that of trastuzumab ADC.

[0434] Example 20 Evaluation of in vitro liver microsome stability

[0435] Each incubation system contains phosphate buffer (PBS, pH 7.4), liver microsomal protein, substrate (acetonitrile solution of the sample to be tested) and NADPH, and is incubated in a 37°C water bath. The same volume of ice-cold acetonitrile is added after 0, 5, 15, 30 and 60 minutes of reaction to terminate the reaction. Negative controls are incubated with heat-inactivated liver microsomes of the corresponding species. The remaining content of the original substrate is detected by LC / MS / MS.

[0436] Example 21 In vivo pharmacokinetic study of antibody-drug conjugates

[0437] Refer to the method described in Yoko Nagai, et al, Comprehensive Preclinical Pharmacokinetic Evaluations of Trastuzumab Deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, in Cynomolgus Monkeys, Xenobiotica, 2019, 49 (9), 1086-1096, to determine the in vivo metabolic pathways and pharmacokinetic parameters of the antibody-drug conjugate of the present application.

[0438] Example 22 Inhibitory effect of antibody-drug conjugates on JIMT-1 Her2-positive breast cancer xenografts in nude mice

[0439] Prepare JIMT-1 breast cancer cells at a concentration of 2 × 10 7 / ml*0.1ml / mouse, inoculated under sterile conditions under the right armpit of nude mice. After subcutaneous tumor inoculation, wait until the tumor volume reaches 100-300mm 3The animals were randomly divided into 3 groups: model group: vehicle (containing: L-histidine 0.89mg / ml, L-histidine hydrochloride 4.04mg / ml, polysorbate 80 0.3mg / ml, sucrose 90mg / ml), 6 animals; 23C2 Her2-2-DXD group: 1.68mg / kg, qw, iv 6 animals; 23C2 Her2-2-DDDXD group: 1.59mg / kg, qw, iv 6 animals. Tumor volume was measured 2-3 times a week, and the mice were weighed and the data were recorded; the performance of the animals was observed daily.

[0440] The relative weight (RWt) calculation formula is:

[0441]

[0442] Wherein Wt0 is the weight of the animals at the time of administration (i.e., d0), and Wt is the weight of the animals at each measurement.

[0443] The calculation formula of tumor growth inhibition (TGI) is:

[0444]

[0445] TW is the tumor weight of the drug-treated group, and TW0 is the tumor weight of the model group.

[0446] Body weight is shown in Table 15 below:

[0447]

[0448] The efficacy is shown in Table 16 below:

[0449]

[0450] The results showed that both 23C2 Her2-2-DXD and 23C2 Her2-2-DDDXD had significant efficacy in the JIMT-1 mouse xenograft tumor model of human breast cancer cells, and 23C2 Her2-2-DDDXD had a stronger tumor inhibition effect than 23C2 Her2-2-DXD.

[0451] According to the contents disclosed in the present application, although the method of the present application is described according to a preferred embodiment, it is apparent to those skilled in the art that the products, elements, methods, and steps or the order of steps described herein may be changed or re-combined without departing from the concept, spirit, and scope of the present application.

[0452] For the purpose of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because they are disclosed prior to the filing date of the present application. All statements about the dates of these documents or the representation of the contents of these documents are based on the information available to the applicant and do not constitute any admission of the correctness of the dates of these documents or the contents of these documents. Moreover, any reference to these publications in this document does not constitute an admission that the publication becomes part of the common general knowledge in the art in any country. The disclosures of all documents cited herein are incorporated herein by reference to the extent that they provide exemplary, procedural and other details supplementing those described herein.

Claims

1. An antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody portion, L represents a linker portion, U represents a cytotoxic drug portion, and n is an integer or decimal selected from 1 to 10, wherein, U is a camptothecin-type topoisomerase I inhibitor, and the L portion and / or the U portion has a deuterated modification.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein U is selected from SN-38, a SN-38 derivative, exatecan, or an exatecan derivative.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein the antibody-drug conjugate comprises the structure shown in the following formula III:

4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein the antibody-drug conjugate comprises the structure shown in the following formula IV: in, R1 is selected from hydrogen or deuterium.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein the antibody-drug conjugate comprises the structure shown in the following formula IV-1:

6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein the antibody-drug conjugate comprises the structure shown in the following formula V: in, R1 and R2 are independently selected from hydrogen or deuterium, Furthermore, the left succinimide terminal of the structure is the linking site to the antibody portion, and the right carbonyl terminal is the linking site to the cytotoxic drug portion.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 6, wherein the antibody-drug conjugate comprises a structure represented by the following formula V-1, V-2, V-3 or V-4: Furthermore, the structure represented by V-1, V-2, V-3 or V-4 is linked to the antibody portion at the left succinimide terminal and is linked to the cytotoxic drug portion at the right carbonyl terminal.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, wherein the antibody-drug conjugate comprises the structure shown in the following formula VI: in, R1 and R2 are each independently selected from hydrogen or deuterium.

9. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 8, wherein the formula VI is the structure shown in the following VI-1, VI-2, VI-3 or VI-4:

10. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 9, wherein the Ab can specifically bind to a tumor antigen selected from HER2, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1 or PD-L1.

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