Antibody drug conjugate

Abstract

Provided is an antibody drug conjugate, specifically comprising a therapeutic antibody moiety, an intermediate linker moiety and a cytotoxic drug moiety which are linked. The therapeutic antibody moiety is an antibody against a HER2 target. The cytotoxic drug moiety is a camptothecin topoisomerase I inhibitor. The cytotoxic drug moiety or the linker-cytotoxic drug moiety is modified by means of deuterium substitution. The antibody drug conjugate can be used for the prevention or treatment of cancers.

Description

Antibody Drug Conjugates Technical Field

[0001] The present application relates to an antibody-drug conjugate comprising a linked therapeutic antibody portion, an intermediate linker portion, and a cytotoxic drug portion. The present application also relates to the use of the antibody-drug conjugate in the preparation of a drug for preventing and treating cancer. Background Art

[0002] Antibody-drug conjugates (ADCs) are a class of drugs that combine the high specificity of therapeutic antibodies with the high cytotoxic activity of cytotoxic drugs. The therapeutic antibody and cytotoxic drug are connected via a linker. Currently, at least eight ADCs are marketed worldwide. The antibodies of brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin target CD30, CD79b, and Nectin-4, respectively; trastuzumab emtansine and trastuzumab deruxtecan target HER2; gemtuzumab ozogamicin and inotuzumab ozogamicin target CD33 and CD22, respectively; and sacituzumab govitecan targets TROP2. As the cytotoxic agent, brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin utilize microtubule-targeting auristatins; trastuzumab emtansine utilizes microtubule-targeting maytansinoids; gemtuzumab ozogamicin and inotuzumab ozogamicin utilize DNA-targeting calicheamicins; and the newly launched trastuzumab deruxtecan and sacituzumab govitecan both utilize camptothecins. As the intermediate linker, trastuzumab emtansine utilizes a non-cleavable linker, while the remaining seven ADCs utilize cleavable linkers.

[0003] Camptothecin (CPT) analogs and derivatives exert their antitumor activity by binding to topoisomerase I, demonstrating significant activity against a wide range of tumor types. To overcome CPT's poor water solubility, researchers have synthesized a variety of CPT derivatives, including irinotecan hydrochloride (CPT-11), a water-soluble prodrug approved for the treatment of metastatic colorectal cancer. However, CPT-11 requires carboxylesterase catalysis to convert into its active form, SN-38 (Formula I), in vivo, a conversion efficiency that is extremely low. SN38 itself is difficult to drug due to its poor solubility. Exatecan (Formula II) (generic name: exatecan), another water-soluble CPT derivative, was previously explored as an antitumor drug, but development was discontinued in 2004. Exatecan does not require enzyme activation and exhibits stronger topoisomerase I inhibitory activity than SN-38, the active form of irinotecan.

[0004]

[0005] 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 high-efficiency and low-toxic ADC drugs that can target more indications.

[0006] 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, specifically involving 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 the antibody portion, L represents the linker portion, U represents the 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 tumor antigens (including tumor-specific antigens and tumor-associated antigens), and the tumor antigen can be selected from any target for tumor prevention or treatment known in the art, for example, 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 changes, additions or deletions 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, wherein the first antigen-binding fragment is a scFv comprising a VH and a VL, wherein the VH has a K30 mutation and / or the VL has an F53 mutation. In some embodiments, the amino acid at position 30 in the VH sequence is mutated from K to an acidic amino acid, such as E. In some embodiments, the amino acid at position 53 in the VL sequence is mutated from F to a neutral or basic amino acid, such as Y, A, or R. For example, the scFv may comprise or be a sequence having a K30 mutation and / or an F53 mutation in the amino acid sequence set forth 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 an 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 comprising the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, the light chain CDR2, and the light chain CDR3 comprising the amino acid sequences of SEQ ID NOs: 30, 34, and 32, respectively; 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 being an 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 comprising the amino acid sequences shown in SEQ ID NOs: 43, 28, and 29, 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: 30, 31, and 32, respectively.

[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 comprise an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 41 and 42, respectively;

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

[0025] EVQLVESGGGLVQPGGSLRLSCAASGFNIX2DTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, X2 is K or E;

[0026] The sequence shown in SEQ ID NO: 42 is:

[0027] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASX1LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, X1 is F or Y.

[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 being an scFv, the first antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the amino acid sequences shown in SEQ ID NOs: 35 and 36, respectively.

[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 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 an scFv, and the VH and VL of the first antigen-binding fragment being arranged from N-terminus to C-terminus in the following order: VH-linker-VL.

[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, and the second antigen-binding fragment is Fab.

[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 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 respectively comprise the amino acid sequences shown in SEQ ID NOs: 45, 46 and 47, 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: 48, 49 and 50.

[0032] 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 a Fab and 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: 37 and 38, respectively.

[0033] 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.

[0034] Table S3 Antibody portion of exemplary antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof

[0035]

[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.CL, CH1, CH2, or CH3, or ii.Fc.

[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 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.

[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 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 or has not been modified; preferably, the modification of the CH3 or Fc is, for example, a substitution of amino acids 435 and / or 436 according to the Kabat numbering system.

[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 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 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.

[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 a constant region operably linked to the first antigen-binding fragment and / or the second antigen-binding fragment, wherein 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. The constant region can be derived from a 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.

[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 an immunoglobulin functional domain or skeleton, such as Fc, 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 be modified 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.

[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 comprises a backbone operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment; in some embodiments, the backbone is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide; in some embodiments, the dimeric Fc has modifications; in some embodiments, the dimeric Fc has H435R and / or Y436F, and the modifications can be in either the first Fc polypeptide or 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; 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.

[0043] 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.

[0044] 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.

[0045] Table S4 Antibody portion of exemplary antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof

[0046]

[0047]

[0048]

[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. The linker portion L of the present application can be linked to the antibody portion by any method known in the art, preferably the linker portion is linked 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 linked to the antibody portion via a sulfhydryl group.

[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 cytotoxic drug portion U is coupled to the antibody portion Ab via a linker portion L, which can 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 can be a low pH-dependent degradation type (including a hydrazone bond, a carbonate bond, etc.), a proteolytic type (including a peptidyl bond), or a high glutathione concentration-dependent degradation type (including a disulfide bond), etc.; in other embodiments, the linker portion of the present application is a non-cleavable linker, for example, it can be a maleimidocaproyl group, etc.

[0051] 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 conjugated to one or more cytotoxic drug portions U. The cytotoxic drug can be selected from, for example, alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, and platinums, etc. Preferred cytotoxic drugs are microtubule inhibitor cytotoxic drugs (including maytansines and auristatins) or DNA-acting cytotoxic drugs (including calicheamicins, duocarmycins, PBDs (pyrrolobenzodiazepines), topoisomerase I inhibitors, etc.).

[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 topoisomerase I inhibitor.

[0053] 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.

[0054] 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, an SN-38 derivative, exatecan, or an exatecan derivative.

[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 are deuterated. In some embodiments, n is an integer or decimal selected from 2 to 10, such as an integer or decimal from 2 to 9, an integer or decimal from 2 to 8, an integer or decimal from 3 to 9, an integer or decimal from 3 to 8, an integer or decimal from 4 to 9, an integer or decimal from 4 to 8, an integer or decimal from 5 to 9, or an integer or decimal from 5 to 8.

[0056] 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.

[0057] 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:

[0058]

[0059] 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:

[0060]

[0061] wherein R1 is selected from hydrogen (H) or deuterium (D).

[0062] 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:

[0063]

[0064] 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:

[0065]

[0066] wherein R1 and R2 are independently selected from hydrogen (H) or deuterium (D),

[0067] 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.

[0068] 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 to the cytotoxic drug portion at the right carbonyl terminal:

[0069]

[0070] 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:

[0071]

[0072] in,

[0073] R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0074] 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:

[0075]

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

[0077]

[0078] in,

[0079] Ab represents an antibody portion comprising a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment being an scFv 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, 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 comprising a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment being an scFv 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, 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 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 comprising a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment being an scFv 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, 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 decimal selected from 1 to 10.

[0096] In a specific embodiment, the present application provides an antibody drug conjugate having a structure 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 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, comprising 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 conjugates of the present application or pharmaceutically acceptable salts or solvates thereof can be used to prevent or treat HER2-positive cancers, HER2-negative cancers (including triple-negative breast cancer), and cancers that show HER2 expression as IHC2+ according to immunohistochemistry.

[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 linking 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 linking an antibody to the intermediate compound:

[0118]

[0119] 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 linking a drug to an antibody via the linker:

[0120]

[0121] wherein R1 and R2 are independently selected from hydrogen (H) or deuterium (D),

[0122] 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.

[0123] 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 linking a drug to an antibody via the linker, wherein the structures shown in formulas V-1 to V-4 are respectively linked to the antibody portion at the left succinimide end and to the cytotoxic drug portion at the right carbonyl end:

[0124]

[0125] In some aspects, the present application provides a compound having a structure shown in the following formula IV(a):

[0126]

[0127] wherein R1 is selected from hydrogen (H) or deuterium (D).

[0128] In some aspects, the present application provides a compound having a structure shown in the following formula IV(a)-1 or IV(a)-2:

[0129]

[0130] In some aspects, the present application provides a compound having a structure shown in the following formula III(a):

[0131]

[0132] The present application provides antibody-drug conjugates or pharmaceutically acceptable salts or solvates 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

[0133] Figure 1 shows the killing rate of BT474 tumor cells by anti-HER2 bispecific antibodies (Expi HER2-1, Expi HER2-2, 23C2 HER2-1, and 23C2 HER2-2) and the combination of trastuzumab and pertuzumab;

[0134] Figure 2 shows the killing rate of NCI-N87 tumor cells by anti-HER2 bispecific antibody, trastuzumab, T-DM1, and the combination of trastuzumab and pertuzumab;

[0135] Figure 3 shows the killing rates of JIMT-1 tumor cells by anti-HER2 bispecific antibody, trastuzumab, T-DM1, and the combination of trastuzumab and pertuzumab;

[0136] FIG4 shows the results of the proliferation inhibition of BT474 tumor cells by anti-HER2 bispecific antibody, trastuzumab, and the combination of trastuzumab and pertuzumab;

[0137] FIG5 shows the effects of anti-HER2 bispecific antibodies, PBS vehicle control, and the combined use of trastuzumab + pertuzumab (Per+Tra) on the changes in tumor volume in the gastric cancer N87 mouse xenograft model;

[0138] FIG6 shows the effects of anti-HER2 bispecific antibody, PBS vehicle control, and the combined use of trastuzumab + pertuzumab on the body weight changes of gastric cancer N87 mice xenografts;

[0139] Figure 7 shows the structures of some exemplary anti-HER2 bispecific antibodies; wherein a dimeric Fc is depicted with one chain shown in black (first Fc polypeptide) and another chain shown in gray (second Fc polypeptide), one antigen-binding domain (first antigen-binding fragment) is shown as hatched, and the other antigen-binding domain (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;

[0140] FIG8 shows the endocytic activity of different antibody-drug conjugates (monocytic antibody-DDDXD and bispecific antibody-DDDXD) in NCI-N87 tumor cells;

[0141] Figure 9 shows the endocytic activity of different antibody-drug conjugates (monocytic antibody-DDDXD and bispecific antibody-DDDXD) in SK-BR-3 tumor cells.

[0142] Unless otherwise indicated, 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 its corresponding commodity or its active ingredient.

[0143] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom 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 an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0144] The term "optionally" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes both the occurrence of the event or situation and the non-occurrence of the event or situation. A group "optionally substituted" means that the group may be substituted or unsubstituted, for example, an ethyl group "optionally" substituted with a halogen means that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (such as CH2CH2F), polysubstituted (such as CHFCH2F, CH2CHF2, etc.) or fully 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 and / or cannot be synthesized will be introduced.

[0145] 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.

[0146] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition on each occurrence is independent. Thus, for example, if a group is substituted with two R's, each R has an independent alternative.

[0147] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a covalent bond.

[0148] 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.

[0149] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0150] The term "hydroxy" refers to an -OH group.

[0151] The term "cyano" refers to a -CN group.

[0152] The term "mercapto" refers to a -SH group.

[0153] The term "amino" refers to a -NH2 group.

[0154] The term "nitro" refers to a -NO2 group.

[0155] The term "alkyl" refers to a group of the formula C n H 2n+1The alkyl group may be straight-chain or branched. For example, the term "C1-6 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.

[0156] The term "alkoxy" refers to an -O-alkyl group.

[0157] The term "alkylamino" refers to an -NH-alkyl group.

[0158] The term "dialkylamino" refers to -N(alkyl)2.

[0159] The term "alkylsulfonyl" refers to an -SO2-alkyl group.

[0160] The term "alkylthio" refers to an -S-alkyl group.

[0161] The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. Non-limiting examples of alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.

[0162] The term "alkynyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH3), 2-propynyl (-CH2-C≡CH), 1,3-butadiynyl (-C≡C≡CH), and the like.

[0163] 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, and the like.

[0164] 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.

[0165] The term "heterocyclic radical" refers to a non-aromatic ring that is fully saturated or partially undersaturated (but not fully unsaturated heteroaromatic) and can exist as a monocycle, a bridged ring or a spirocycle. Unless otherwise indicated, the heterocycle is typically a 3 to 7 ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulphur, oxygen and / or nitrogen. The limiting examples of heterocyclic radicals include but are not limited to oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl etc.

[0166] 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 heterocycloalkyl groups include, but are not limited to, oxirane, thioethane, and cyclonitroethane; non-limiting examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, and thietanyl; examples of 5-membered heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, and tetrahydropyrazolyl; examples of 6-membered heterocycloalkyl groups 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 heterocycloalkyl groups include, but are not limited to, azepanyl, oxetanyl, and thiepanyl. Monocyclic heterocycloalkyl groups having 5 or 6 ring atoms are preferred.

[0167] 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 can 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.

[0168] The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, with the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryl groups have single 4 to 8-membered rings, especially 5 to 8-membered rings, or multiple fused rings containing 6 to 14, especially 6 to 10, ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.

[0169] "Derivative": A compound formed by replacing atoms or atomic groups in the parent compound molecule with other atoms or atomic groups is called a derivative of the parent compound.

[0170] Unless otherwise specified, any atom in a labeled synthetic compound herein may represent any stable isotope of that atom. Unless otherwise specified, when a position in a structure is defined as H, i.e., hydrogen (H-1), that position contains only naturally occurring isotopes. Similarly, unless otherwise specified, when a position in a structure is defined as D, i.e., deuterium (H-2), that position contains an isotope at least 3340 times greater than the naturally occurring isotope (0.015%) (i.e., at least 50.1% deuterium isotope). When one or more positions in the structure of a labeled synthetic compound are defined as D, i.e., deuterium (H-2), the content of the compound represented by that 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%, or at least 99.5%. The deuteration rate of a labeled synthetic compound as used herein 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 in a labeled synthetic compound as used herein may be at least 3500-fold (52.5%), at least 4000-fold (60%), at least 4500-fold (67.5%), at least 5000-fold (75%), at least 5500-fold (82.5%), at least 6000-fold (90%), at least 6333.3-fold (95%), at least 6466.7-fold (97%), at least 6566.7-fold (98.5%), at least 6600-fold (99%), or at least 6633.3-fold (99.5%). Isotopologues as used herein refer to compounds that differ in chemical structure only in their isotopic composition. The compounds synthesized and labeled herein have identical chemical structures, varying only in isotopic composition. Therefore, the compounds synthesized and labeled herein containing deuterium at specific positions will also contain very low amounts of hydrogen isotopologues at those positions. The amount of hydrogen isotopologues at a particular position in the compounds synthesized and labeled herein 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 the deuterium isotope. However, as previously mentioned, the total amount of hydrogen isotopologues at a particular position will be less than 49.9%. The total amount of hydrogen isotopologues at a particular position in the compounds synthesized and labeled herein will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1%, or 0.5%.

[0171] In this application, any atom not designated as deuterium is present at its natural isotopic abundance.

[0172] The term "treating" means administering a compound or formulation described herein to prevent, ameliorate, or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) preventing the occurrence of a disease or disease state in a mammal, particularly when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state; (ii) inhibiting the disease or disease state, i.e., curbing its development; and (iii) relieving the disease or disease state, i.e., causing the disease or disease state to regress.

[0173] The term "therapeutically effective amount" means an amount of a compound of the present invention 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 a compound of the present invention 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 this disclosure.

[0174] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, 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.

[0175] 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.

[0176] The term "solvate" refers to an association of a compound with solvent molecules.

[0177] 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.

[0178] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0179] 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 prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. The specific example of a proton tautomer is the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions by reorganization of some bonding electrons.

[0180] 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 (e.g., bispecific antibodies), multifunctional antibodies, and antibody fragments, so long as they possess the desired biological activity.

[0181] The term "humanized antibody" refers to an antibody that comprises CDR regions derived from non-human antibodies, while the remaining portions of the antibody molecule are derived from one or more human antibodies.

[0182] 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 substituted with an amino acid different from that of 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".

[0183] The term "CDR" (complementarity determining region), also known as "hypervariable region", refers to each of the regions of an antibody variable domain that are highly variable in sequence and / or form structurally defined loops. A natural four-chain antibody typically contains six CDRs, three in the heavy chain variable region and three in the light chain variable region.

[0184] The term "variable region" refers to an antibody structural unit composed of two pairs of polypeptide chains, each pair having a heavy chain and a light chain, and the N-terminal domain of each chain defines a region of about 100 to 110 or more amino acids primarily responsible for antigen recognition as the variable region.

[0185] The term "Fab" refers to the constant domain (CL) of the light chain 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.

[0186] 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.

[0187] The term "ECD" refers to the extracellular domain. HER receptors are receptor protein tyrosine kinases that belong to the human epidermal growth factor receptor (HER) family and include EGFR, HER2, HER3, and HER4 receptors. HER2 receptors typically 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 phosphorylated tyrosine residues. The HER2 extracellular domain consists of four domains: ECD1, ECD2, ECD3, and ECD4.

[0188] The term "antibody portion" refers to the antibody portion of 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.

[0189] The term "linker portion" refers to the portion of the antibody-drug conjugate that connects the antibody portion to the cytotoxic drug portion and can be cleavable or non-cleavable. A cleavable linker refers to a linker that can be cleaved in the target cell, thereby releasing the cytotoxic drug.

[0190] The term "cytotoxic drug moiety" refers to the cytotoxic drug portion of the antibody-drug conjugate. In certain specific schemes, it is connected to the intermediate linker portion through a functional group. In tumor cells, cytotoxic drug molecules will be released, thereby exerting an anti-tumor effect.

[0191] The term "trastuzumab": 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.

[0192] 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.

[0193] 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.

[0194] The term "cancer" refers to the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0195] 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.

[0196] As used herein, unless otherwise stated, the terms "comprise, comprise, and comprising" or their equivalents (contain, contains, containing, include, include, including, 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.

[0197] Unless otherwise indicated herein, all numbers used herein expressing amounts of ingredients, measurements, or reaction conditions are to be understood as being modified in all instances by the term "about." When used in conjunction with a percentage, the term "about" can mean, for example, ±0.1%, preferably ±0.05%, and more preferably ±0.01%.

[0198] 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.

[0199] In this 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 (e.g. www.ncbi.nlm.nih.gov), such as BLASTp or BLASTn with default settings. DETAILED DESCRIPTION

[0200] For the sake of clarity, the present application is further illustrated 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.

[0201] The trastuzumab and pertuzumab used in the examples of this application were prepared according to conventional antibody methods, starting with vector construction, transfection into eukaryotic cells, and subsequent purification and expression. The sequence of trastuzumab is referenced in WHO DRUG INFORMATION INN RL78, and the sequence of pertuzumab is referenced in the Examples section of WO0100245. DS-8201 is the active ingredient in the commercially available formulation Enhertu from Daiichi Sankyo Co., Ltd., and its structure is identical to trastuzumab-DXD prepared in this application (see Example 15 for its structure).

[0202] Example 1 Construction, expression and purification of anti-Her2 scFv-Fc and its variants

[0203] When constructing anti-Her2 scFv-Fc, the Fc portion was human IgG1, and the variable region sequence of the anti-Her2 arm was 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), the amino acid sequence of which is as follows:

[0204]

[0205] In addition, point mutations were constructed on the anti-Her2 scFv-Fc sequence to construct the following variants:

[0206] anti-Her2-scFv-VL-F53Y-Fc (SEQ ID NO: 3): Derived from wild-type anti-Her2 scFv-Fc, with an F53Y mutation in the VL region; its amino acid sequence is as follows:

[0207]

[0208] anti-Her2-scFv-VL-F53A-Fc (SEQ ID NO: 5): derived from wild-type anti-Her2 scFv-Fc, with the F53A mutation in the VL region; its amino acid sequence is as follows:

[0209]

[0210]

[0211] anti-Her2-scFv-VL-F53R-Fc (SEQ ID NO: 7): derived from wild-type anti-Her2 scFv-Fc, with the F53R mutation in the VL region; its amino acid sequence is as follows:

[0212]

[0213] 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:

[0214]

[0215] 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 vectors of anti-Her2 scFv-Fc or its variants were co-transfected into ExpiCHO cells (CHO-S, Thermo company) using an expression kit (Thermo Fisher, catalog number A29133). The cells were cultured in an incubator at 37°C in a humidified atmosphere containing 8% CO2 in ExpiCHO expression medium on an orbital shaker platform rotating at 130 rpm. The culture supernatant was collected and protein purification was performed using protein A magnetic beads (Genscript, catalog number L00273). Protein concentration was measured by UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).

[0216] Table 1: Sequence information of anti-Her2 scFv-Fc and its variants

[0217]

[0218] Example 2 Aggregation Validation of Anti-Her2 scFv-Fc and Its Variants and Binding Detection to Human Her2 Antigen

[0219] The expressed and purified anti-Her2 scFv-Fc and its variants were assayed for affinity to human Her2 protein using Biacore T200 (GE). The experimental procedure is described as follows:

[0220] A certain amount of anti-Her2 scFv-Fc or its variants were captured using an anti-hIgG-coupled chip. Human HER2 protein (Sino Biological, catalog number 10004-H08H) was then passed over the chip surface. The reaction signal was detected in real time using a Biacore T200 to generate binding and dissociation curves. The buffer used in the experiment was Biacore Universal Buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄·12H₂O, 1.8 mM KH₂PO₄, 0.05% surfactant P20 (GE, catalog number BR-1000-54), pH 7.4). Anti-hIgG (captured using the Human Antibody Capture Kit, GE Healthcare, catalog number 29-2346-00) was coupled to a CM5 chip surface, resulting in a reaction rate of approximately 9000 RU. Anti-Her2 scFv-Fc or its variants were captured, resulting in a reaction rate of approximately 200 RU. Signals from the interaction between anti-Her2 scFv-Fc or its variants and different concentrations of human HER2 protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) were then measured. The flow cell was operated at a flow rate of 50 μl / min, with an association time of 240 s, a dissociation time of 1400 s, and a 60 s regeneration with 3 M MgCl2 (GE Healthcare) to stabilize the baseline. Results were calculated using the affinity and kinetics 1:1 binding model using the biacore evaluation software. The affinities of anti-Her2 scFv-Fc or its variants for the antigen human HER2 protein are shown in Table 2. Compared to anti-Her2 scFv-Fc (KD = 0.77 nM), the F53A mutation significantly affected the binding affinity of the variant anti-Her2-scFv-VL-F53A-Fc (KD = 1.26 nM) to the antigen human Her2 protein. The binding affinity of the variant anti-Her2-scFv-VL-F53Y-Fc (KD = 0.8 nM) to the antigen human Her2 protein was similar to that of the anti-Her2 scFv-Fc (KD = 0.77 nM). The KD of the variant anti-Her2-scFv-VH-K30E-Fc to the antigen human Her2 protein was 0.54 nM. This indicates that the introduction of the F53Y and K30E mutations does not reduce the binding affinity to the antigen human Her2 protein.

[0221] The components of anti-Her2 scFv-Fc or its variants were separated using a gel chromatography column, and the components were eluted in descending order of molecular weight. The column used was an ACQUITY UPLC Protein BEH SEC Column. A 1.7 μm, 4.6 x 300 mm gel chromatography column was used at 25°C. The mobile phase consisted of 50 mmol / L phosphate buffer-200 mmol / L sodium chloride, pH 7.0 (weigh 2.33 g of sodium dihydrogen phosphate dihydrate, 12.53 g of disodium hydrogen phosphate dodecahydrate, and 11.69 g of sodium chloride, add approximately 800 ml of ultrapure water, stir until fully dissolved, add ultrapure water to 1000 ml, mix thoroughly, and filter through a 0.22 μm filter). The sample was diluted to 10 mg / ml with the mobile phase as the test solution. Accurately aspirate 2 μl and inject it into the liquid chromatograph (if the sample concentration is lower than 10 mg / ml, adjust the injection volume to 20 μg of protein). Detection was performed at a wavelength of 280 nm. The flow rate was 0.30 ml / min, and the elution was isocratic for 15 min. Data were processed and quantitatively analyzed using the area normalization method. The peak area percentages of aggregates, immunoglobulin monomers, and low-molecular-weight impurities were calculated, with aggregates preceding the main peak, immunoglobulin monomers representing the main peak, and low-molecular-weight impurities following the main peak representing the main peak. The ratios of the main peak to aggregate content for anti-Her2 scFv-Fc and its variants are shown in Table 2. The variants anti-Her2-scFv-VL-F53Y-Fc and anti-Her2-scFv-VH-K30E-Fc significantly reduced aggregate content, from 6.31% in anti-Her2 scFv-Fc to 4.94% and 3.39%, respectively.

[0222] Table 2: Aggregate content and binding affinity of anti-Her2 scFv-Fc and its variants to the antigen Her2

[0223]

[0224] Example 3 Construction, expression, and purification of anti-Her2 bispecific antibodies

[0225] An anti-Her2 bispecific antibody was generated as a human IgG1 using Knobs-into-holes (Ridgway, et al., 1996) Fc engineering. The H435R and Y436F mutations in the Fc region of one heavy chain (Jendeberg et al., 1997) were engineered to reduce the Fc affinity for Protein A, facilitating the removal of homodimers formed during bispecific antibody assembly during Protein A affinity purification (Patent US5945311A). As can be seen from Example 2, 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 example, the antigen-binding domain of one anti-Her2 arm of the anti-Her2 bispecific antibody is in the form of an scFv (VH-linker-VL structure), and the variable region sequence contains the K30E mutation (b-anti-Her2-scFv-VH-K30E-Fc, SEQ ID NO: 21), the F53Y mutation (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 example, the antigen-binding domain of the other anti-Her2 arm of the anti-Her2 bispecific antibody is in Fab format, comprising anti-Her2-domain2-HC-Fc (SEQ ID NO: 13) and anti-Her2-domain2-LC (SEQ ID NO: 15). Additionally, an anti-Her2 bispecific antibody constructed using a scFv (VH-linker-VL structure or VL-linker-VH structure) without mutations served as a control.

[0226] Table 3: Sequence information of anti-Her2 bispecific antibodies

[0227]

[0228]

[0229]

[0230] The DNA sequences of anti-Her2 bispecific antibodies (SEQ ID NOs: 12, 14 and 16) were synthesized and cloned into pcDNA3.1 expression vectors. 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) were co-transfected into ExpiCHO cells at a transfection ratio of 1:1:1.5 using the high-yield expression system (Cat. No.: MIR 6270). The transfection density was 6×10 6 cells / ml. The culture medium is Expression medium (catalog number MIR 6200, manufacturer Mirus). Continuous culture was performed until the 10th day after transfection, and the cell culture supernatant was collected by centrifugation. Protein purification was performed using protein A magnetic beads (Genscript, catalog number L00273). Protein concentration was measured using a 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.

[0231] Example 4 Preparation and Verification of Fucose Knockout Bispecific Antibodies

[0232] By knocking out the fucose expression-related gene FUT8, the interaction between IgG1 and FcgRIIIa can be improved, 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 (designated as 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) was used to co-transfect 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 (catalog number MIR 6200, manufacturer Mirus). Continuously 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). 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 CHO FUT8- / - 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.

[0233] Anti-Her2 bispecific antibody samples expressed in CHO FUT8- / - cells and CHO-S cells were treated with the GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, Mass., USA) to release N-glycans from the protein. The N-glycans were then labeled and separated by chromatographic column. The N-glycan structures and contents were determined using an FLR detector (Waters, Milford, Mass., USA). The glycoform content ratios are shown in Table 4. The proportion of fucose-free in the normal anti-Her2 bispecific antibody Expi HER2-2 was 21.85%, while the proportion of fucose-free in the anti-Her2 bispecific antibody 23C2 HER2-2 expressed in FUT8-knockout CHO-S cells was 99.40%.

[0234] Table 4: Glycoform content analysis of anti-Her2 bispecific antibodies

[0235]

[0236]

[0237] Example 5 Aggregation Verification of Bispecific Antibodies

[0238] 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.

[0239] The anti-HER2 bispecific antibody was separated using a gel chromatography column to verify the aggregate content. The components were eluted in descending order of molecular weight. The column is ACQUITY UPLC Protein BEH SEC Column. A 1.7 μm, 4.6 x 300 mm gel chromatography column was used at 25°C. The mobile phase consisted of 50 mmol / L phosphate buffer-200 mmol / L sodium chloride, pH 7.0 (weigh 2.33 g of sodium dihydrogen phosphate dihydrate, 12.53 g of disodium hydrogen phosphate dodecahydrate, and 11.69 g of sodium chloride, add approximately 800 ml of ultrapure water, stir until fully dissolved, add ultrapure water to 1000 ml, mix thoroughly, and filter through a 0.22 μm filter). The sample was diluted to 10 mg / ml with the mobile phase as the test solution. Accurately aspirate 2 μl and inject it into the liquid chromatograph (if the sample concentration is lower than 10 mg / ml, adjust the injection volume to 20 μg of protein). Detection was performed at a wavelength of 280 nm. The flow rate was 0.30 ml / min, and the elution was isocratic for 15 min. Data were processed and 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 peaks before the main peak were aggregates, the main peak was immunoglobulin monomers, and the peaks after the main peak were low molecular weight impurities.

[0240] Table 5: Aggregate content of anti-Her2 bispecific antibodies

[0241] Sample name Aggregate Monomer Low molecular weight fragment 23C2 Her2-2 8.51% 91.02% 0.47% 23C2 Her2-1 19.55% 80.05% 0.40%

[0242] 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 23C2 Her2-1 without mutations, the aggregate content of 23C2 Her2-2 could be reduced to 8.51% (Table 5).

[0243] Example 6 Antigen Binding Detection of Anti-Her2 Bispecific Antibodies

[0244] The expressed and purified anti-Her2 bispecific antibody was assayed for affinity between the test molecule and the HER2 protein using Biacore T200 (GE). The experimental procedure is described as follows:

[0245] A certain amount of anti-Her2 bispecific antibody was captured using an anti-hIgG-coupled chip. Human Her2 (Sino Biological, catalog number 10004-H08H) was then passed over the chip surface. The reaction signal was detected in real time using a Biacore T200 to generate binding and dissociation curves. The buffer used in the experiment was Biacore Universal Buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P20, pH 7.4). Anti-hIgG (captured using a human antibody capture kit, GE, catalog number 29-2346-00) coupled to a CM5 chip surface achieved a response of approximately 9000 RU. After capturing the anti-Her2 bispecific antibody, the response was approximately 200 RU. Signals from the interaction between the anti-Her2 bispecific antibody and various concentrations of Her2 protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) were then measured. The flow cell was operated at a flow rate of 50 μl / min, with an association time of 240 s and a dissociation time of 1400 s. The baseline was stabilized after regeneration with 3 M MgCl2 (GE) for 60 s.

[0246] Results were calculated using biacore evaluation software. The binding affinities of the anti-Her2 bispecific antibody 23C2 Her2-2 and the control antibodies trastuzumab and pertuzumab for the Her2 antigen are shown in Table 6. The KD for binding of the anti-Her2 bispecific antibody 23C2 Her2-2 to the Her2 antigen was 6.11E-10 M, the KD for binding of trastuzumab to the Her2 antigen was 1.22E-09 M, and the KD for binding of pertuzumab to the Her2 antigen was 2.35E-09 M. The anti-Her2 bispecific antibody 23C2 Her2-2 has a higher affinity for the Her2 antigen than trastuzumab and pertuzumab.

[0247] Table 6: Affinity of anti-Her2 bispecific antibodies for human Her2 antigen

[0248] ka(1 / Ms)kd(1 / s)KD(M)23C2 Her2-21.79E+051.09E-046.11E-10Trastuzumab control sample1.73E+052.12E-041.22E-09Pertuzumab control sample1.15E+052.69E-042.35E-09

[0249] Example 7 Antigen Binding Detection of Anti-Her2 Bispecific Antibodies

[0250] 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 was determined by Biacore T200 (GE).

[0251] The affinity test results of the anti-Her2 bispecific antibody 23C2 Her2-1 for 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 for the antigen human Her2 protein.

[0252] Table 7: Affinity of anti-Her2 bispecific antibodies for human Her2 antigen

[0253] Sample Name 23C2 Her2-1 Antigen Her2 KD 4.02nM

[0254] Example 8 Killing of Her2-positive target cells BT474 by anti-Her2 bispecific antibodies

[0255] Human PBMCs (peripheral blood mononuclear cells) were used to provide NK cells to study the killing effect of the anti-Her2 bispecific antibody on target cells (BT474Her2+++, source: Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences). The in vitro activity of the anti-Her2 bispecific antibody was evaluated based on the EC50 value.

[0256] 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 into a 96-well cell culture plate (eppendorf, cat. no. 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 added to the above 96-well cell culture plate at 50 μl per well. Human PBMCs were adjusted to a cell density of 1.5 × 10 cells / ml using 1640 experimental medium. 6cells / ml, 100μl per well. Set up the drug administration group (target cells + effector cells + antibody), target cell group (BT474 cells), effector cell group (human PBMC), target cell + effector cell group, blank control group (culture medium), lysate control group, and target cell maximum release group (target cells + lysate), with an effector-target ratio of 10:1. 45 minutes before the test, 20μl / well lysate (Promega, catalog number G182A) was added to the target cell maximum release group and lysate control group. Use after 45 minutes. The cell lysis rate was detected by cytotox 96 nonradioactive cytotoxicity assay kit (Promega, G1780).

[0257] 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%

[0258] FIG1 shows the killing rate of the anti-Her2 bispecific antibody against BT474Her2+++ tumor cells. The ADCC-enhanced anti-Her2 bispecific antibodies (represented as 23C2 HER2-1 and 23C2 HER2-2 in Figure 1) were superior to the combined trastuzumab + pertuzumab and CHO-S-expressed anti-Her2 bispecific antibodies (Expi HER2-1 and Expi HER2-2) in killing BT474 tumor cells. The EC50 of the trastuzumab + pertuzumab (1:1) combination was 8.627 ng / ml, while the EC50 of Expi HER2-1 was 38.05 ng / ml. Expi HER2-2 was superior to Expi HER2-1, with an EC50 of 35.17 ng / ml. The EC50 of 23C2 HER2-1 with ADCC-enhanced was 4.728 ng / ml, while the ADCC-enhanced 23C2 HER2-2 was superior to the ADCC-enhanced 23C2. HER2-1, with an EC50 of 3.658 ng / ml.

[0259] Example 9 Killing of Her2-positive target cells NCI-N87 by anti-Her2 bispecific antibodies

[0260] Human PBMCs (peripheral blood mononuclear cells) were used to provide NK cells to study the killing effect of the anti-Her2 bispecific antibody on target cells (NCI-N87Her2++, source: Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences). The in vitro activity of the anti-Her2 bispecific antibody was evaluated based on the EC50 size.

[0261] 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, 50 μl per well was inoculated into a 96-well cell culture plate (eppendorf, cat. no. 0030730199). Different concentrations of anti-Her2 bispecific antibody or control drug (8.1 nM, 2.7 nM, 0.9 nM, 0.3 nM, 0.1 nM, 0.03 nM, 0.01 nM, 0.003 nM, 0.001 nM, 0.0004 nM) were prepared using experimental culture medium and added to the above 96-well cell culture plate at 50 μl per well. Human PBMCs were adjusted to a cell density of 1.5 × 10 cells / ml using experimental culture medium. 6 cells / ml, 100μl per well. Set up the drug administration group (target cells + effector cells + 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), lysate control group, and target cell maximum release group (target cells + lysate), with an effector-target ratio of 10:1. 45 minutes before the test, 20μl / well lysate (Promega, catalog number G182A) was added 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 + pertuzumab (1:1), and Expi HER2-1 were used as control drugs.

[0262] 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%

[0263] Figure 2 shows the killing rate of NCI-N87 tumor cells by anti-Her2 bispecific antibodies. The ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 demonstrated superior killing of NCI-N87 tumor cells compared to the combination of trastuzumab + pertuzumab, trastuzumab, T-DM1, and Expi HER2-1. The EC50 for ADCC-enhanced 23C2 HER2-2 was 0.02447 nM, the EC50 for the combination of trastuzumab + pertuzumab was 0.08267 nM, the EC50 for Expi HER2-1 was 0.1048 nM, the EC50 for T-DM1 was 0.07392 nM, and the EC50 for trastuzumab was 0.07468 nM.

[0264] Example 10 Killing of Trastuzumab-resistant JIMT-1 cells by anti-Her2 bispecific antibody

[0265] Human PBMCs (peripheral blood mononuclear cells) were used to provide NK cells to study the cytotoxic effect of the anti-Her2 bispecific antibody on target cells (JIMT-1, source: AddexBio, Catalog #: C0006005). The in vitro activity of the anti-Her2 bispecific antibody was evaluated based on the EC50 value.

[0266] 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, 50 μl per well was inoculated into a 96-well cell culture plate (eppendorf, cat. no. 0030730199). Different concentrations of anti-Her2 bispecific antibody or control drug (8.1 nM, 2.7 nM, 0.9 nM, 0.3 nM, 0.1 nM, 0.03 nM, 0.01 nM, 0.003 nM, 0.001 nM, 0.0004 nM) were prepared using experimental culture medium and added to the above 96-well cell culture plate at 50 μl per well. Human PBMCs were adjusted to a cell density of 1.5 × 10 cells / ml using experimental culture medium. 6 cells / ml, 100μl per well. Set up the drug administration group (target cells + effector cells + antibody or control drug), target cell group (BT474 cells), effector cell group (human PBMC), target cell + effector cell group, blank control group (culture medium), lysate control group, and target cell maximum release group (target cells + lysate), with an effector-target ratio of 20:1. 45 minutes before the test, 20μl / well lysate (Promega, catalog number G182A) was added to the target cell maximum release group and lysate control group. Use 45 minutes later The cell lysis rate was determined using the Cytotox 96 nonradioactive cytotoxicity assay (Promega, G1780). Trastuzumab, T-DM1, trastuzumab plus pertuzumab (1:1), and Expi HER2-1 were used as control drugs.

[0267] 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%

[0268] Figure 3 shows the killing rate of JIMT-1 tumor cells by anti-Her2 bispecific antibodies. The ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 demonstrated superior killing of JIMT-1 tumor cells compared to the combination of trastuzumab + pertuzumab, trastuzumab, T-DM1, and Expi HER2-1. The EC50 for ADCC-enhanced 23C2 HER2-2 was 0.01006 nM, compared to the EC50 for the combination of trastuzumab + pertuzumab, 0.06727 nM, 0.08066 nM for Expi HER2-1, 0.08357 nM for T-DM1, and 0.07443 nM for trastuzumab. Furthermore, the anti-Her2 bispecific antibody 23C2 HER2-2 exhibited a higher cell lysis rate.

[0269] Example 11 Inhibition of proliferation of BT474 Her2+++ tumor cells by anti-Her2 bispecific antibodies

[0270] 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, GIBCO, catalog number 10099-141). Then, a 1:1 gradient dilution was performed 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, and 0.00625 μg / ml). BT474 Her2+++ cells in the logarithmic growth phase were obtained and the density was adjusted to 1×10 5Cells / ml were plated, 100 μl was added to each well, and a blank well without cells was set up as a control. The above-mentioned gradient diluted samples were added, 50 μl per well. Cultured in an incubator at 37°C and 5% CO2 for 5 days. Discard the culture medium, add CCK-8 (Japan Dojin Chemical, product number: CK04) working solution, 100 μl per well, incubate for 4 to 5 hours, and then place in a microplate reader (manufacturer Thermo, model: VarioskanFlash), with 630 nm as the reference wavelength, read and record the absorbance value of the well plate at a wavelength of 450 nm. Calculate the tumor cell proliferation inhibition rate.

[0271] The results are shown in Figure 4. The ADCC-enhanced anti-Her2 bispecific antibody 23C2 Her2-2 had an inhibition rate of 78.38% on BT474 tumor cell proliferation, which was superior to the inhibition rate of trastuzumab (54.12%) and the inhibition rate of trastuzumab + pertuzumab combined (53.7%).

[0272] Example 12 Inhibitory Effect of Anti-Her2 Bispecific Antibody on NCI-N87 Her2++ Gastric Cancer Xenografts in Nude Mice

[0273] The in vivo efficacy of the anti-Her2 bispecific antibody was evaluated in a mouse xenograft model using NCI-N87 Her2++ gastric cancer cells (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences). NCI-N87 Her2++ gastric cancer cells were prepared at a concentration of 5×10 7 The solution was inoculated under sterile conditions into the right axilla of nude mice (from Changzhou Cavens Laboratory Animal Co., Ltd., male, 14-17 g, housed in SPF grade). The diameter of the transplanted tumor in the nude mice was measured with a vernier caliper. The tumor was then grown to 100-250 mm. 3 The animals were then divided into 5 groups:

[0274] Group 1: Control

[0275] Group 2: Expi Her2-1, 10 mg / kg

[0276] Group 3: 23C2 Her2-2, 5 mg / kg

[0277] Group 4: 23C2 Her2-2, 10 mg / kg

[0278] Group 5: Per+Tra (trastuzumab + pertuzumab), 5 mg / kg + 5 mg / kg

[0279] Each treatment group received the corresponding dose via intravenous injection twice weekly for approximately three weeks (six doses). Except for Group 5, where the combination therapy group received both drugs at a volume of 5 ml / kg each, all other groups received a 10 ml / kg dose. Group 1 also received 10 ml / kg of PBS (Hyclone; Catalog No. sh30256.01) intravenously. For the combination therapy group, trastuzumab was administered at least 30 minutes after pertuzumab.

[0280] The anti-tumor effect of the test substance was dynamically observed by measuring tumor diameter. 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.

[0281] Detection indicators:

[0282] Tumor volume (TV) is calculated as follows: TV = 1 / 2 × a × b 2 , where a and b represent the length and width of the tumor, respectively.

[0283] In this experiment, drug administration began on day 0 and was repeated six times (days 0, 3, 7, 10, 14, and 17). No animals died by day 21. The average body weight of mice in each group showed an increasing trend (Figure 6). No significant toxic effects were observed.

[0284] By d21, the effects of each test sample 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 xenograft tumors in nude mice are shown in Table 8 and Figure 5. The 23C2 Her2-2 group (10 mg / kg) had better inhibition on NCI-N87 gastric cancer xenograft tumors in nude mice than Expi Her2-1 (10 mg / kg) and Per+Tra combination group (5 mg / kg+5 mg / kg).

[0285] Table 8: Effects of anti-HER2 dual antibodies on the volume of NCI-N87 gastric cancer xenografts in nude mice (mean ± SD)

[0286]

[0287] Example 13 Synthesis of MC-GGFG-DXD

[0288]

[0289] Step 1 A5 (Synthesis of ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl acetate)

[0290] Weigh 20g of SM5 (N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycine) into a 1L three-necked flask, add 300ml of tetrahydrofuran and 100ml of toluene, stir thoroughly, then add 30g of lead tetraacetate and 5.4g of pyridine. Heat to 65°C and react for 4 hours. Cool to room temperature and filter out the solids. Concentrate the organic phase to dryness at 40°C. Add 300ml of ethyl acetate and 300ml of water to the concentrated organic phase. Stir for 20 minutes. Separate the ethyl acetate phase. Add 100ml of saturated sodium chloride solution to the ethyl acetate phase and stir for 20 minutes. Separate the ethyl acetate phase and concentrate. Silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) yields 13g of A5. Yield: 63%. ESI-MS: m / z = 391.1 [M+Na] + .

[0291] Step 2: Synthesis of B5 ([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]benzyl acetate)

[0292] Weigh 1.0g of A5 into a 250ml single-necked flask, add 15ml of DME (ethylene glycol dimethyl ether), add 0.897g of benzyl glycolate, and cool to 0°C in an ice-water bath. Prepare a solution of 0.27ml of water and 0.108g of NaOH. Add the prepared NaOH solution to the reaction mixture. React at 0°C for 1 hour, then add 0.078g of glacial acetic acid. Add 100ml of water and 100ml of ethyl acetate, stir at room temperature for 20 minutes, separate the organic phase, and concentrate to dryness. Silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) yields 0.68g of B5. Yield: 53%. ESI-MS: m / z = 497.1 [M+Na] + .

[0293] Step 3: Synthesis of C5 ([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid)

[0294] Weigh 0.68 g of B5 into a 250 ml hydrogenation flask, add 20 ml of ethanol and 10 ml of ethyl acetate, and add 0.34 g of wet palladium on carbon (10% palladium content). Bring hydrogen gas into the flask using a hydrogen balloon. React at room temperature for 1 hour. Filter the palladium on carbon through celite. Concentrate the filtrate to dryness at 40°C. 425 mg of C5 is obtained. Yield: 77%. ESI-MS: m / z = 407.1 [M+Na] + .

[0295] 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]glycineamide)

[0296] 50 mg of SM4 (isatecan mesylate) and 50 mg of C5 were weighed into a 100 ml single-necked flask. 2 ml of DMF (N,N-dimethylformamide) was added, and the temperature was cooled to 0°C. 54 mg of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and 30 mg of DIEA (N,N-diisopropylethylamine) were added. The mixture was allowed to return to room temperature and allowed to react for 3 hours. 100 ml of dichloromethane and 100 ml of water were added. The mixture was stirred for 20 minutes. The organic phase was separated and concentrated to dryness. Silica gel column chromatography (dichloromethane:methanol = 10:1) yielded 88 mg of D5. The yield was 84%. ESI-MS: m / z = 802.4 [M+H]. + .

[0297] 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]glycineamide)

[0298] Weigh 78 mg of D5 into a 100 ml single-necked flask, add 4 ml of tetrahydrofuran, cool to 0°C, and add 78 mg of ethylenediamine. Return to room temperature and react for 5 hours. Concentrate the reaction mixture to dryness at 40°C to obtain 55 mg of E5. Yield: 98%. ESI-MS: m / z = 580.3 [M+H] + Step 6: Synthesis of MC-GGFG-DXD

[0299] Weigh 55 mg of E5 into a 100 mg single-necked flask, add 2 ml of N,N-dimethylformamide, add 48 mg of MC-GGF-OH (maleimidocaproylglycylglycylphenylalanine), cool to 0°C, add 54 mg of HATU, and add 30 mg of DIEA. Return to room temperature and react for 1 hour. Add 100 ml of ethyl acetate and 100 ml of water, stir for 20 minutes, separate the organic phase, and concentrate to dryness at 40°C. Silica gel column chromatography (ethyl acetate:methanol = 10:1) yields 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).

[0300] Example 14 Synthesis of Deuterated MC-GGFG-DXD (MC-GGFG-DDDXD), Deuterated DXD (DDDXD) and DXD

[0301]

[0302] Step 1 Synthesis of Intermediate A

[0303] Under nitrogen, 80g of ethyl diazoacetate was added to a 3L single-necked flask. 800ml of dichloromethane and 800ml of a 1% deuterated acetic acid solution (8g of deuterated acetic acid dissolved in 800ml of deuterated water) were added. The reaction mixture was stirred at room temperature under light-shielding conditions for 75 hours. The organic phase was separated and collected, and the aqueous phase was extracted twice with dichloromethane (200ml x 2). The organic phases were combined. The organic phase was washed with 200ml of deuterated water, dried over anhydrous sodium sulfate, filtered to remove the sodium sulfate, and the filtrate was concentrated to dryness under reduced pressure at 20°C to obtain 49.25g of Intermediate A. The yield was 66%. 1 H NMR (500MHz, CDCl3) δ4.27 (q, J=7.1Hz, 1H), 1.31 (t, J=7.1Hz, 2H).

[0304] Step 2 Synthesis of Intermediate B

[0305] Weigh 50g of N-Fmoc-glycyl-glycine into 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, raise the temperature to 80°C, and continue reacting for 3 hours. Cool to room temperature, filter, and wash the filter cake with 250ml of ethyl acetate. Concentrate the filtrate to dryness. Dissolve the mixture in 330ml of dichloromethane and 670ml of ethyl acetate to obtain an organic phase. Wash the organic phase three times with 30% potassium bicarbonate solution (500ml x 3). Dry the organic phase over anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure, dissolve it in 100ml of dichloromethane, then add 100ml of n-hexane. Stir at room temperature until a solid precipitates. Then, add 300ml of a mixture of n-hexane and dichloromethane (n-hexane:dichloromethane = 1:1) and stir overnight. The filter cake was filtered and dried 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] + .

[0306] Step 3 Synthesis of Intermediate C

[0307] Weigh 78g of intermediate B into a 3000ml single-necked flask, add 800ml of dichloromethane, and add 45g of compound A. Place the 3000ml single-necked flask in an ice-water bath and cool 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. Bring to room temperature, add 800ml of water and stir. Separate the liquid and collect the organic phase. Extract the aqueous phase with 400ml of dichloromethane. Combine the organic phases. Wash the organic phase once with 800ml of saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate 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] + .

[0308] Step 4: Synthesis of Compound D

[0309] 31.2 g of compound C was added to 270 mL of deuterated methanol and 70 mL of heavy water, stirred in an ice bath, and then 5.5 g of NaOH was added. The mixture was brought to room temperature and stirred overnight. 300 mL of ethyl acetate and 300 mL of water were added to the reaction solution for extraction. 20 mL of glacial acetic acid was added to the aqueous layer to adjust the pH to 2-3. A solid precipitated and was filtered to obtain 20.3 g of compound D. Yield: 69.8%. LCMS (ESI) m / z: 409.08 [M+Na] + .

[0310] Step 5 Synthesis of Compound E

[0311] Weigh 2.0 g of exatecan mesylate dihydrate and 1.63 g of compound D into a 100 ml round-bottom flask. Add 40 ml of N,N-dimethylformamide and stir. Cool to 0°C. Add 2.0 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 1.82 g of N,N-diisopropylethylamine in sequence. React at 0°C for 3 h. Pour the reaction solution into 120 ml of ice water. Stir for 1 h. Filter. Dissolve the filter cake in dichloromethane. Column chromatography (100 g of 100-200 mesh silica gel, dichloromethane:methanol = 30:1, 2 L) yields 2.6 g of compound E. Yield: 92%. LCMS (ESI) m / z: 804.84 [M+H] + .

[0312] Step 6 Preparation of Compound F

[0313] Weigh 0.38 g of 1,8-diazabicyclo[5.4.0]undec-7-ene into a 100 ml round-bottom flask. Add 20 ml of tetrahydrofuran to the flask and stir. Cool to 0°C. Weigh 2.0 g of compound E and prepare a solution with 20 ml of tetrahydrofuran. Slowly add the prepared compound E solution to the 100 ml round-bottom flask. Allow the reaction mixture to warm to room temperature and react for 3 hours. Filter under nitrogen. 1.45 g of compound F is obtained. Yield: 98%. LCMS (ESI) m / z: 582.39 [M+H]. + .

[0314] Step 7 Preparation of Compound H

[0315] Weigh 1.00 g of compound F and 0.97 g of compound G into a 100 ml round-bottom flask, then add 10 ml of N,N-dimethylformamide. Cool to -20°C, then add 0.34 g of 1-hydroxybenzotriazole and 0.49 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Allow to react at -20°C for 3 h. Add 20 ml of dichloromethane and 20 ml of water to the reaction mixture, stir for 0.5 h, let stand, separate, and collect the organic phase. Dry the organic phase over anhydrous sodium sulfate and filter. Concentrate the filtrate to dryness under reduced pressure. Silica gel column chromatography (dichloromethane:methanol = 15:1) yields 400 mg of compound H. Yield: approximately 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).

[0316]

[0317]

[0318] Step 1 Synthesis of Intermediate I

[0319] 0.85 g of compound A was added to 9 ml of deuterated methanol and 1 ml of heavy water, stirred under ice bath, and then 0.32 g of sodium hydroxide was added. The mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure at 40°C to obtain compound I, which was used directly in the next step without purification.

[0320] Step 2 Synthesis of compound J(DDDXD)

[0321] 0.30 g of exatecan mesylate dihydrate and 0.056 g of intermediate I were added to 3 ml of N,N-dimethylformamide. Stirring was continued under ice-cooling. Subsequently, 0.32 g of 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate and 0.22 g of N,N-diisopropylethylamine were added and stirred at room temperature for 4 h. The reaction mixture was purified by liquid phase chromatography to obtain 0.18 g of compound J. Yield: 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).

[0322] In addition, DXD was prepared according to the method disclosed in Example 76 in the specification of patent WO2014057687.

[0323]

[0324] Example 15 Preparation of Antibody Drug Conjugates

[0325] Reagents:

[0326] Solution A: pH 7.4 PBS buffer

[0327] Solution B: 10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution

[0328] Solution C: DMSO (dimethyl sulfoxide)

[0329] Solution D: Histidine buffer (containing 0.89 mg / ml L-histidine and 4.04 mg / ml L-histidine hydrochloride monohydrate)

[0330] Solution E: 700 mg / ml sucrose solution (prepared using solution D)

[0331] Solution F: 20 mg / ml Tween 80 (prepared with Solution D)

[0332] Antibodies: Trastuzumab, 23C2 Her2-2

[0333] Linker-payload (linker-cytotoxic drug part): MC-GGFG-DXD, MC-GGFG-DDDXD

[0334] Table 9. Experimental conditions and groups:

[0335]

[0336] Experimental process:

[0337] 1. Antibody replacement

[0338] a. Fully wet the 30KD ultrafiltration centrifuge tube with solution A;

[0339] b. Replace the antibody into solution A;

[0340] 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).

[0341] 2. Antibody reduction

[0342] a. Calculate the molar amount of antibody, recorded as N1;

[0343] b. Add an appropriate amount of solution B to the antibody solution so that the molar amount of TCEP in the reaction system is N2;

[0344] 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.

[0345] 3. Coupling

[0346] a. Take an appropriate amount of linker-payload and dissolve it in DMSO to a final concentration of 10 mg / ml;

[0347] b. Add DMSO to the antibody solution to make the antibody concentration 5wt%, then add an appropriate amount of linker-payload solution to make the molar concentration

[0348] N3;

[0349] 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.

[0350] 4. Coupling termination

[0351] a. Wet the ultrafiltration centrifuge tube with solution D;

[0352] b. Replace the antibody into solution D, add appropriate amounts of solutions E and F, and adjust the concentrations of sucrose and Tween 80 to 90 mg / ml and 0.3 mg / ml, respectively. Freeze at -80°C.

[0353] Determination of the DAR value (average number of drug linkages per antibody molecule) of antibody-drug conjugates

[0354] DAR values ​​were determined using LC-MS. 50 μg of the prepared ADC sample was added to 1 μl of glycosidase PNGase F (Ruian Biotechnology, China) and incubated at 37°C for 20 hours. The mass spectrometer used in the experiment was a high-resolution Xevo G2-XS (Waters, USA). The sample concentration was adjusted to 5 μM, and direct injection was used to acquire mass spectrometric data in positive ion mode. The acquired native mass spectrometric data were analyzed and processed using UNIFI 1.8.2.169 software (Waters, USA).

[0355] Protein concentration determination of antibody drug conjugates

[0356] Protein concentration was determined using the Lowry method. Trastuzumab and 23C2 Her2-2 were used as standards. The absorbance of the standards and the prepared ADC samples at OD650 was measured using a microplate reader. A standard curve was fitted, and the sample absorbance was subtracted from the standard curve to calculate the protein concentration.

[0357] The following antibody-drug conjugates were prepared and assayed using the above method:

[0358]

[0359] Trastuzumab-DXD, antibody concentration: 4.25 mg / ml, DAR: 7.6.

[0360]

[0361] Trastuzumab-DDDXD, antibody concentration: 4.29 mg / ml, DAR: 7.7.

[0362]

[0363] 23C2 Her2-2-DXD, antibody concentration: 4.35 mg / ml, DAR: 5.7.

[0364]

[0365] 23C2 Her2-2-DDDXD, antibody concentration: 4.16 mg / ml, DAR: 5.8.

[0366] Example 16 Deuterated DXD (DDDXD) in vitro enzyme activity

[0367] 1. Preparation of reagents and materials

[0368] a. Prepare 1% agarose electrophoresis gel;

[0369] b. Prepare gelred dye solution and store it away from light;

[0370] c. Preparation of topoisomerase I working solution: Prepare with ultrapure water and buffer.

[0371] 2. Sample preparation

[0372] a. Redissolve and dilute the compound (DDDXD) with DMSO. The compound concentration starts from 200 μM, and is diluted 10-fold. Set up 5 gradients. 3. Reaction system

[0373] a. Positive control: 15 μl ultrapure water + 2 μl 10× DNA Topoismerasel Buffer + 2 μl 0.1% BSA + 1 μl pBR322 DNA;

[0374] b. Negative control: 14 μl ultrapure water + 2 μl 10× DNA Topoismerasel Buffer + 2 μl 0.1% BSA + 1 μl pBR322 DNA + 1 μl topoisomerase I working solution;

[0375] 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.

[0376] 4. Experimental process

[0377] a. Place the above reaction system in a 37°C water bath for 30 min;

[0378] b. Add 2 μl loading buffer to each tube to terminate the reaction;

[0379] c. Agarose gel electrophoresis at 2-2.5 V / cm for 1.5 h;

[0380] d. After electrophoresis, stain the gel with GelRed in the dark for 1.5 h and take pictures with a gel imager.

[0381] Example 17 Antigen Binding Detection of Antibody Drug Conjugates

[0382] Referring to the method of Example 6, the affinities of Trastuzumab, 23C2 HER2-2, Trastuzumab-DDDXD and 23C2 HER2-2-DDDXD to HER2 protein were measured and are shown in Table 10 below:

[0383] Sample ka(1 / Ms)kd(1 / s)KD(M)Trastuzumab1.23E+051.12E-049.16E-1023C2 HER2-21.73E+056.06E-053.49E-10Trastuzumab-DDDXD1.28E+051.19E-049.25E-1023C2 HER2-2-DDDXD1.28E+053.26E-052.55E-10

[0384] The results showed that anti-Her2 bispecific antibody had stronger antigen binding activity than trastuzumab, and anti-Her2 bispecific antibody ADC had stronger antigen binding activity than trastuzumab ADC.

[0385] Example 18 Endocytosis Experiment of Antibody Drug Conjugate

[0386] 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, 20 μl / 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 40 μg / ml, labeled S1, and then 3-fold serial dilution was performed to obtain the corresponding samples S1-S9. In addition, DS-8201 and control IgG1 (a non-HER2 target specific IgG1; Sino Biological, Cat. No. HG1K) and DDDXD conjugate IgG1-DDDXD were used as controls. The serially diluted sample solution and the labeled endocytosis reagent (sartorius, 90564) were added to the cell plate, 20 μl was added to each well, and incubated at 37°C for 15 minutes. The 96-well cell culture plate was removed, and the two cells were added at 20 μl / well respectively. The cell culture plate was placed at 37°C and incubated for another 2 hours. The 96-well cell culture plate was removed and placed on a flow cytometer to measure the signal intensity.

[0387] The results of the endocytosis experiments on two HER2-positive cells, NCI-N87 and SK-BR-3, are shown in Figures 8 and 9. The results show that the endocytosis of the anti-Her2 dual antibody ADC is stronger than that of the trastuzumab ADC.

[0388] Example 19 Cellular Activity of Deuterated DXD and Antibody Drug Conjugates

[0389] DXD and DDDXD were pre-diluted to 140,000 ng / ml using culture medium, labeled S1, and then five-fold serial dilutions were performed to obtain the corresponding reference samples S1-S9. The final drug concentration range was 35,000 ng / ml to 0.0896 ng / ml, for 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 were plated at 500 μl per well and 100 μl was added to each well. A blank well without cells was set up as a control. 50 μl of the above two samples were added in gradient dilutions to each well. Culture was carried out in a 37°C, 5% CO2 incubator for 5 days. The culture medium was discarded and CCK-8 (Dojin Chemical, Japan, Product No.: CK04) working solution was added to each well at 100 μl. After incubation for 4 to 5 hours, the plate was placed in a microplate reader (Thermo, Model: VarioskanFlash) and 630 nm was used as the reference wavelength. The absorbance value of the well plate at a wavelength of 450 nm was read and recorded. The tumor cell proliferation inhibition rate was calculated.

[0390] The results of the NCI-N87 cell experiment are shown in Table 11 below:

[0391] Sample IC 50 (nM)DXD4.45DDDXD4.01

[0392] DDDXD exhibited stronger tumor cell proliferation inhibitory activity than DXD.

[0393] The test antibody and the antibody drug conjugate prepared in Example 15 were pre-diluted to 20 μg / ml using culture medium, labeled S1, and then diluted five-fold 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 of each was adjusted to 2×10 4Cells were plated at 50 cells / ml, with 100 μl added to each well. A blank well without cells was set up as a control. Serially diluted samples were added at 50 μl per well. The cells were incubated at 37°C in a 5% CO2 incubator. The culture medium was discarded and 100 μl of CTG detection solution (Promega, Catalog No. G7572) was added to each well. After incubation for 10 minutes to develop color, the cells were placed on a multi-function plate reader (Thermo, Model: VarioskanFlash) and the chemiluminescence value was read. The tumor cell proliferation inhibition rate was calculated.

[0394] The results of the NCI-N87 cell experiment are shown in Table 12 below:

[0395] Sample inhibition rate% Trastuzumab 34.51 Trastuzumab-DDDXD 70.81 23C2 HER2-2 75.73 23C2 HER2-2-DDDXD 90.08

[0396] The results of the BT474 cell experiment are shown in Table 13 below:

[0397] Sample inhibition rate% Trastuzumab 67.91 Trastuzumab-DDDXD 47.82 23C2 HER2-2 75.56 23C2 HER2-2-DDDXD 73.76

[0398] The results of the SK-BR-3 cell experiment are shown in Table 14 below:

[0399] Sample inhibition rate% Trastuzumab 20.36 Trastuzumab-DDDXD 67.15 23C2 HER2-2 62.89 23C2 HER2-2-DDDXD 75.56

[0400] From the above results, it can be seen that the cell killing ability of anti-Her2 bispecific ADC is better than that of trastuzumab ADC.

[0401] Example 20 In vitro liver microsome stability evaluation

[0402] Each incubation system contained phosphate buffered saline (PBS, pH 7.4), liver microsomal protein, substrate (test sample in acetonitrile), and NADPH. Incubation was performed in a 37°C water bath. The reaction was terminated by adding an equal volume of ice-cold acetonitrile after 0, 5, 15, 30, and 60 minutes. Negative controls were incubated with heat-inactivated liver microsomes from the corresponding species. The remaining unchanged substrate content was determined by LC / MS / MS.

[0403] Example 21 In vivo pharmacokinetic experiments of antibody-drug conjugates

[0404] The in vivo metabolic pathways and pharmacokinetic parameters of the antibody-drug conjugate of the present application were determined with reference 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.

[0405] Example 22 Inhibitory Effect of Antibody Drug Conjugates on JIMT-1 Her2-Positive Breast Cancer Xenografts in Nude Mice

[0406] 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 transplantation, wait until the tumor volume reaches 100-300mm 3 Animals were randomly divided into three groups: the model group (n=6) received vehicle (containing: L-histidine 0.89 mg / ml, L-histidine hydrochloride 4.04 mg / ml, polysorbate 80 0.3 mg / ml, sucrose 90 mg / ml); the 23C2 Her2-2-DXD group (n=6) received 1.68 mg / kg, qw, i.v.; and the 23C2 Her2-2-DDDXD group (n=6) received 1.59 mg / kg, qw, i.v. Tumor volume was measured 2-3 times per week, and the mice were weighed and weighed, and the data were recorded. Animal performance was observed daily.

[0407] The formula for calculating relative weight (RWt) is:

[0408]

[0409] 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.

[0410] The calculation formula for tumor growth inhibition (TGI) is:

[0411]

[0412] TW is the tumor weight of the drug-treated group, and TW0 is the tumor weight of the model group.

[0413] Body weight is shown in Table 15 below:

[0414]

[0415] The efficacy is shown in Table 16 below:

[0416]

[0417] The results showed that both 23C2 Her2-2-DXD and 23C2 Her2-2-DDDXD had significant efficacy in the human breast cancer cell JIMT-1 mouse xenograft tumor model, and 23C2 Her2-2-DDDXD had a stronger tumor inhibition effect than 23C2 Her2-2-DXD.

[0418] 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.

[0419] For the purposes of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements regarding 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 as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications herein does not constitute an admission that such publications become 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 supplementary to 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, in, 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 any one of claims 1 to 2, wherein the antibody-drug conjugate comprises a structure shown in the following formula III or formula IV: or In Formula IV, R 1 Selected from hydrogen (H) or deuterium (D).

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 VI: in, R 1 , R 2 are independently selected from hydrogen (H) or deuterium (D).

5. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein the formula VI is the structure shown in the following VI-1, VI-2, VI-3 or VI-4: or 6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein Ab is an antibody that can specifically bind to HER2.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 6, wherein the 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 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: 27, 28 and 29, 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: 30, 34 and 32, respectively.

8. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein 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, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise the amino acid sequences shown in 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 shown in SEQ ID NOs: 30, 31 and 32, respectively.

9. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein the first antigen-binding fragment is selected from: 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 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 comprise an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 41 and 42, respectively.

10. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 7 to 9, wherein the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

36.

11. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 9 to 10, wherein 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.

12. The antibody-drug conjugate according to any one of claims 6 to 11, 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.

13. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 12, wherein the second 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, wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise 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 comprise the amino acid sequences shown in SEQ ID NOs: 48, 49 and 50, respectively.

14. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 12 to 13, wherein the second 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 comprise the amino acid sequences of SEQ ID NOs: 37 and 38, respectively.

15. The antibody-drug conjugate according to any one of claims 6 to 14, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab comprises an immunoglobulin domain operably linked to the first antigen-binding fragment and / or the second antigen-binding fragment, the immunoglobulin domain comprising: i. CL, CH1, CH2, or CH3, or ii. Fc.

16. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 15, wherein the CL, CH1, CH2, CH3, and Fc are derived from CL, CH1, CH2, CH3, and Fc of human IgG, respectively.

17. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 16, wherein the CL, CH1, CH2, CH3 or Fc has a modification or has no modification; preferably, the modification of CH3 or Fc is a substitution of the 435th and / or 436th amino acid according to the Kabat numbering system.

18. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 17, 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.

19. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 6 to 18, wherein the antibody portion Ab is a bivalent bispecific antibody comprising: a heavy chain with a sequence as shown in SEQ ID NO: 11, a heavy chain with a sequence as shown in SEQ ID NO: 13, and a light chain with a sequence as shown in SEQ ID NO:

15.

20. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the antibody-drug conjugate has a structure shown in the following formula VII: in, 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 shown in 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 shown in SEQ ID NOs: 30, 31 and 32, respectively; 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 shown in 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 shown in SEQ ID NOs: 45, 46 and 47, respectively. NOs: amino acid sequences shown in 48, 49 and 50, n is an integer or decimal selected from 1 to 10, R 1 , R 2 are independently selected from hydrogen (H) or deuterium (D).

21. The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 20, wherein Formula VII is the structure shown in VII-1, VII-2, VII-3 or VII-4 below: or 22. Use of the antibody-drug conjugate according to any one of claims 1 to 21 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for preventing or treating cancer; preferably, the cancer is HER2-positive cancer, HER2-negative cancer, and cancer in which HER2 expression is shown as IHC2+ according to immunohistochemical detection.

23. The linker-drug intermediate compound of the structure shown in Formula VI: in, R 1 , R 2 are independently selected from hydrogen (H) or deuterium (D).

24. The linker-drug intermediate compound according to claim 23, in, Formula VI is the structure shown in the following VI-1, VI-2, VI-3 or VI-4: or 25. A compound having a structure represented by the following formula IV(a) or formula III(a): in, R 1 is selected from hydrogen (H) or deuterium (D); or