Antibody-drug conjugates

Abstract

Provided is an antibody-drug conjugate comprising a linked therapeutic antibody moiety, an intermediate linker moiety, and a cytotoxic drug moiety, wherein the therapeutic antibody moiety is a HER2-targeting antibody, the cytotoxic drug moiety is a camptothecin-class topoisomerase I inhibitor, and the cytotoxic drug moiety or the linker-cytotoxic drug moiety is modified with deuterium substitution. The antibody-drug conjugate can be used for the prevention or treatment of cancer. TIFF2023537051000091.tif76170

Description

[Technical Field]

[0001] This application relates to an antibody-drug conjugate comprising a linked therapeutic antibody portion, an intermediate linker portion, and a cytotoxic drug portion. This application further relates to the use of the antibody-drug conjugate in the manufacture of drugs for the prevention and treatment of cancer. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are drugs that combine the high specificity of a therapeutic antibody with the high killing activity of a cytotoxic drug, in which the therapeutic antibody portion and the cytotoxic drug portion are linked via an intermediate linker. Currently, at least eight ADC drugs are marketed worldwide. Of these, 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 The antibody portion of govitecan targets TROP2. As for the cytotoxic drug portion, brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin employ auristatins toxin molecules that act on microtubules, trastuzumab emtansine employs maytansinoid toxin molecules that act on microtubules, gemtuzumab ozogamicin and inotuzumab ozogamicin employ calicheamicins toxin molecules that act on DNA, and the newly launched trastuzumab deruxtecan and sacituzumab govitecan both employ camptothecin toxin molecules. As for the intermediate linker portion, trastuzumab emtansine employs a non-cleavable linker, while the other seven ADC drugs all employ cleavable linkers.

[0003] Camptothecin (CPT) analogs and derivatives exert antitumor activity by binding to topoisomerase I and exhibit clear activity against many types of tumors. To overcome the drawback of poor water solubility of CPT, some researchers have synthesized various CPT derivatives. Among them, irinotecan hydrochloride (CPT-11) is a water-soluble prodrug and is approved for use in the treatment of metastatic colorectal cancer. However, CPT-11 must be catalyzed by carboxylesterase to be converted into its active form SN-38 (Formula I) in vivo. This conversion is extremely inefficient, and SN38 itself has poor solubility and thus is difficult to become a drug. Exatecan (Formula II) (generic name: exatecan) is another water-soluble CPT derivative, and its development as an antitumor drug has been carried out but ended in 2004. This does not require enzymatic activation, and exatecan has stronger inhibitory activity against topoisomerase I than SN-38, which is the origin of the efficacy of irinotecan. [Chemical formula]

[0004] ADC drugs combine two advantages: the high efficacy of cytotoxic small molecules and the high selectivity of antibodies for specific tumor cells. Currently, it is still necessary to develop effective and low-toxic ADC drugs for more indications. [Summary of the Invention]

[0005] In one aspect of the present application, an antibody-drug conjugate containing deuterium modification or a pharmaceutically acceptable salt or solvate thereof is provided, specifically related to the deuterium modification of the linker or the cytotoxic drug moiety.

[0006] In one aspect of the present application, an antibody-drug conjugate having the general formula Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein Ab represents an antibody moiety, L represents a linker moiety, U represents a cytotoxic drug moiety, and n is selected from an integer or a decimal between 1 and 10.

[0007] In one embodiment of the present invention, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the Ab (antibody portion) can specifically bind to a tumor antigen (including tumor-specific antigens and tumor-associated antigens), and the tumor antigen may be selected from any tumor-preventing or therapeutic targets known in the art, such as HER2, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1, PD-L1, etc.

[0008] In some embodiments of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, where Ab (the antibody portion) may be modified, for example, by alteration, increase, or decrease of one or more amino acids.

[0009] In some embodiments of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, where the antibody portion Ab is an antibody that can specifically bind to HER2.

[0010] In some embodiments, the antibody portion Ab of the antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is trastuzumab and has the sequence shown in Table S1 below. [Table 1]

[0011] In some embodiments, the antibody portion Ab of the antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is pertuzumab and has the sequence shown in Table S2 below. [Table 2]

[0012] In some embodiments of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, and the first antigen-binding fragment is an scFv comprising VH and VL, wherein VH has a K30 mutation and / or 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, for example, 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, for example, Y, A, or R. For example, scFv may include, or may not include, a sequence having the K30 mutation and / or F53 mutation in the amino acid sequence shown in SEQ ID NO: 1.

[0013] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment being scFv, the first antigen-binding fragment comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 27, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs: 30, 34, and 32, respectively, wherein the sequence shown in SEQ ID NOs: 27 is GFNIX2DTYIH, where X2 is K or E, and the sequence shown in SEQ ID NOs: 34 is SASX1LYS, where X1 is F or Y.

[0014] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment being scFv, and the first antigen-binding fragment comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 43, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively.

[0015] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, and the first antigen-binding fragment is scFv and selected from the following. i. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region each comprise the amino acid sequence shown in SEQ ID NOs. 41 and 42, or ii. The first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each comprising an amino acid sequence having at least 80% identity with the amino acid sequences shown in SEQ ID NOs. 41 and 42. Here, the sequence shown in sequence number 41 is EVQLVESGGGLVQPGGSLRLSCAASGFNIX2DTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, where X2 is K or E. The sequence shown in sequence number 42 is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASX1LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK, where X1 is F or Y.

[0016] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment is scFv, and the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region respectively, It contains the amino acid sequences shown in SEQ ID NOs. 35 and 36.

[0017] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, the first antigen-binding fragment is scFv, and the VH and VL of the first antigen-binding fragment are arranged in the order VH-linker-VL from the N-terminus to the C-terminus.

[0018] In one embodiment of the present invention, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab further comprises a monovalent second antigen-binding fragment that specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, and the second antigen-binding fragment is Fab.

[0019] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab further comprises a monovalent second antigen-binding fragment that specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, the second antigen-binding fragment being Fab and comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 48, 49, and 50, respectively.

[0020] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab further comprises a monovalent second antigen-binding fragment that specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, the second antigen-binding fragment being Fab and 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: 37 and 38, respectively.

[0021] In some specific embodiments, the antibody portion Ab of the antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is as shown in Table S3. [Table 3]

[0022] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, the immunoglobulin functional domain comprising i.CL, CH1, CH2, or CH3, or ii.Fc.

[0023] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, the immunoglobulin functional domain comprising i. 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.

[0024] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, the immunoglobulin functional domain comprising i. CL, CH1, CH2, or CH3, or ii. Fc, wherein the CL, CH1, CH2, CH3, or Fc may or may not be modified, preferably the modification of the CH3 or Fc being, for example, a substitution of an amino acid at positions 435 and / or 436 based on Kabat numbering.

[0025] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, the immunoglobulin functional domain comprising i.CL, CH1, CH2, or CH3, or ii.Fc, wherein Fc is a dimer Fc comprising a first Fc polypeptide and a second Fc polypeptide, the first antigen-binding fragment being operably linked to the first Fc polypeptide, and the second antigen-binding fragment being operably linked to the second Fc polypeptide.

[0026] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a constant region operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, the constant region may be a constant region of the native sequence of an immunoglobulin or a mutated constant region, for example, one or more native or mutated CL, CH1, CH2 and / or CH3 functional domains, in some examples, the functional domains are operably linked in a manner common to the art, the constant region may be derived from a constant region of a human immunoglobulin, for example, from IgG1, IgG2, IgG3 or IgG4, and in some examples, the constant region may have modifications to improve its ability to mediate effector function.

[0027] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab is an immunoglobulin functional domain or framework operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, for example, comprising Fc, the term Fc comprising a native sequence Fc region and a variant Fc region, where Fc may be, for example, a human Fc derived from IgG1, IgG2, IgG3, or IgG4, and Fc may have modifications to improve its ability to mediate effector function, for example, in some embodiments, the framework is knob-into-hole The modifications include H435R, Y436F, and defucoselation, and in some embodiments, the knob-into-hole mutation sites of the framework include, for example, Y349C, T366S, L368A, Y407V, S354C, T366W, etc.

[0028] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab comprises a framework operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, wherein in some embodiments the framework is a dimer Fc comprising a first Fc polypeptide and a second Fc polypeptide, wherein in some embodiments the dimer Fc has modifications, wherein in some embodiments the dimer Fc has H435R and / or Y436F, wherein the modifications are of the first Fc polypeptide and the second Fc polypeptide. It may be located on either polypeptide chain, and in some specific embodiments, the dimer Fc has H435R and / or Y436F, the modification occurring only on one Fc polypeptide and not on the other, and in some embodiments, the dimer Fc is a knob-into-hole mutation site having, for example, Y349C, T366S, L368A, Y407V, S354C, T366W, etc., and in some embodiments, one chain of the dimer Fc has T366W and / or S354C, and the other chain has Y407V, Y349C, T366S or / and L368A.

[0029] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the antibody portion Ab is a bivalent bispecific antibody comprising a heavy chain containing the sequence of SEQ ID NO: 11, a heavy chain containing the sequence of SEQ ID NO: 13, and a light chain containing the sequence of SEQ ID NO: 15.

[0030] In some specific embodiments, the antibody portion Ab of the antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is as shown in Table S4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0031] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, where the cytotoxic drug portion U and the antibody portion Ab are conjugated by a linker portion L. In the present application, the linker portion L can be linked to the antibody portion by any method known in the art, preferably by a mercapto group and / or an amino group. In some more preferred embodiments of the present application, the linker portion is linked to the antibody portion by a mercapto group.

[0032] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, wherein the cytotoxic drug portion U and the antibody portion Ab are conjugated by a linker portion L, the linker portion may be a cleavable linker or a non-cleavable linker, and in some embodiments of the present application, the linker portion is a cleavable linker, for example, a low pH degradable type (including hydrazone bonds, carbonate bonds, etc.), a protease degradable type (including peptide bonds), or a high glutathione concentration degradable type (including disulfide bonds), and in some embodiments of the present application, the linker portion is a non-cleavable linker, for example, a maleimidocaproyl group, etc.

[0033] In one embodiment of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, where the antibody portion Ab is conjugated with one or more cytotoxic drug portions U, the cytotoxic drug may be selected from, for example, alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, platinum compounds, etc. Preferred cytotoxic drugs are microtubule inhibitors (including maytansinoids and auristatins) or DNA-acting cytotoxic drugs (including calichemycins, duocarmycins, PBDs (pyrrolobenzodiazepines), topoisomerase I inhibitors, etc.).

[0034] In some specific embodiments, the cytotoxic drug portion U of an antibody-drug conjugate having the general formula Ab-(LU)n provided herein, or a pharmaceutically acceptable salt or solvate thereof, is a topoisomerase I inhibitor.

[0035] In some specific embodiments, the cytotoxic drug portion U of an antibody-drug conjugate having the general formula Ab-(LU)n provided herein, or a pharmaceutically acceptable salt or solvate thereof, is a camptothecin topoisomerase I inhibitor.

[0036] In some specific preferred embodiments, the cytotoxic drug portion U of an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is selected from SN-38, SN-38 derivatives, exatecan, or exatecan derivatives.

[0037] In some embodiments of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, where Ab represents the antibody moiety, L represents the linker moiety, U represents a camptothecin topoisomerase I inhibitor, and n is selected from an integer or decimal number between 1 and 10, where the L moiety and / or U moiety are deuterium modified. In some embodiments, n is an integer or decimal number between 2 and 10, for example, selected from an integer or decimal number between 2 and 9, an integer or decimal number between 2 and 8, an integer or decimal number between 3 and 9, an integer or decimal number between 3 and 8, an integer or decimal number between 4 and 9, an integer or decimal number between 4 and 8, an integer or decimal number between 5 and 9, or an integer or decimal number between 5 and 8.

[0038] In some embodiments of the present application, an antibody-drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof is provided, where Ab represents the antibody moiety, L represents the linker moiety, U represents a camptothecin-type topoisomerase I inhibitor, and n is selected from an integer or decimal number between 1 and 10, wherein the L moiety and / or U moiety are deuterium-modified, and the cytotoxic drug moiety U is selected from SN-38, SN-38 derivatives, exatecan, or exatecan derivatives.

[0039] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof provided herein, having the general formula Ab-(LU)n, include the structure shown in the following formula III. [ka]

[0040] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof provided herein, having the general formula Ab-(LU)n, include the structure shown in the following formula IV: [ka] In the formula, R1 is selected from hydrogen (H) or deuterium (D).

[0041] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof having the general formula Ab-(LU)n provided herein include the structure shown in the following formulas IV-1 or IV-2. [ka]

[0042] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof provided herein, having the general formula Ab-(LU)n, include the structure shown in the following formula V: [ka] In the formula, R1 and R2 are each independently selected from hydrogen (H) or deuterium (D). Furthermore, the succinimide terminus on the left side of the structure is a binding site to the antibody portion, and the carbonyl terminus on the right side is a binding site to the cytotoxic drug portion.

[0043] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof provided herein, having Ab-(LU)n as the general formula, include structures represented by the following formulas V-1, V-2, V-3, or V-4, wherein the structures represented by formulas V-1 to V-4 are linked to the antibody moiety at the left succinimide terminus and to the cytotoxic drug moiety at the right carbonyl terminus. [ka]

[0044] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof provided herein, having the general formula Ab-(LU)n, include the structure shown in the following formula VI: [ka] In the formula, R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0045] In some embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof having the general formula Ab-(LU)n provided herein include structures represented by the following formulas VI-1, VI-2, VI-3, or VI-4. [ka] [ka] [ka] [ka]

[0046] In some specific embodiments of the present application, an antibody-drug conjugate having the structure shown in formula VII, or a pharmaceutically acceptable salt or solvate thereof, is provided. [ka] Here, Ab represents the antibody portion and comprises a first antigen-binding fragment and a second antigen-binding fragment, wherein the first antigen-binding fragment is scFv and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 43, 28, and 29, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 30, 31, and 32, respectively. The second antigen-binding fragment is Fab and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 45, 46, and 47, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 48, 49, and 50, respectively. n is selected from integers or decimals between 1 and 10. R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0047] In some specific embodiments of the present application, antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof having structures represented by the following formulas VII-1, VII-2, VII-3, or VII-4 are provided. [ka] [ka] [ka] [ka] Here, Ab represents the antibody portion and comprises a first antigen-binding fragment and a second antigen-binding fragment, wherein the first antigen-binding fragment is scFv and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 43, 28, and 29, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 30, 31, and 32, respectively. The second antigen-binding fragment is Fab and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 45, 46, and 47, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 48, 49, and 50, respectively. n is selected from integers or decimals between 1 and 10.

[0048] In a particular embodiment of the present application, an antibody-drug conjugate having the structure shown in formula VII-1, or a pharmaceutically acceptable salt or solvate thereof, is provided. [ka] Here, Ab represents the antibody portion and comprises a first antigen-binding fragment and a second antigen-binding fragment, wherein the first antigen-binding fragment is scFv and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 43, 28, and 29, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 30, 31, and 32, respectively. The second antigen-binding fragment is Fab and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 45, 46, and 47, respectively, and light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences of SEQ ID NOs. 48, 49, and 50, respectively. n is selected from integers or decimals between 1 and 10.

[0049] In a particular embodiment of the present application, an antibody-drug conjugate having the structure shown in formula VII, or a pharmaceutically acceptable salt or solvate thereof, is provided. [ka] Here, Ab is trastuzumab, n is selected from integers or decimals between 1 and 10. R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0050] In a particular embodiment of the present application, an antibody-drug conjugate having the structure shown in formula VII-1, or a pharmaceutically acceptable salt or solvate thereof, is provided. [ka] Here, Ab is trastuzumab, n is selected from integers or decimals between 1 and 10.

[0051] In one embodiment of the present application, a pharmaceutical composition is provided comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0052] In one embodiment of the present application, the use of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a drug for the prevention or treatment of cancer is provided.

[0053] In one embodiment of the present application, the use of a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier in the manufacture of a drug for the prevention or treatment of cancer is provided.

[0054] In one embodiment of the present invention, an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof for the prevention or treatment of cancer is provided.

[0055] One embodiment of the present invention provides a method for treating or preventing cancer, comprising administering to a patient in need a therapeutically effective amount of the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

[0056] In some embodiments, the antibody-drug conjugates of the present invention 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 in which HER2 expression is indicated as IHC2+ by immunohistochemistry.

[0057] In some aspects of this application, a linker-drug intermediate compound having the structure shown in the following formula VI is provided for obtaining an antibody-drug conjugate that links an antibody and an intermediate compound. [ka] In the formula, R1 and R2 are each independently selected from hydrogen (H) or deuterium (D).

[0058] In some aspects of this application, linker-drug intermediate compounds having the structure shown in the following formulas VI-1, VI-2, VI-3, or VI-4 are provided for obtaining antibody-drug conjugates that link an antibody and an intermediate compound. [ka] [ka] [ka] [ka]

[0059] In some aspects of this application, a linker compound having the structure shown in the following formula V is provided for obtaining an antibody-drug conjugate that links a drug and an antibody via a linker. death, [ka] Here, R1 and R2 are each independently selected from hydrogen (H) or deuterium (D). Furthermore, the succinimide terminus on the left side of the structure is a binding site to the antibody portion, and the carbonyl terminus on the right side is a binding site to the cytotoxic drug portion.

[0060] In some embodiments of the present invention, linker compounds having the structures shown in the following formulas V-1, V-2, V-3, or V-4 are provided for obtaining antibody-drug conjugates that link a drug and an antibody via a linker, where the structures shown in formulas V-1 to V-4 are linked to the antibody moiety at the left succinimide terminus and to the cytotoxic drug moiety at the right carbonyl terminus. [ka] [ka] [ka] [ka]

[0061] In some embodiments of this application, a compound having the structure shown in the following formula IV(a) is provided, [ka] In the formula, R1 is selected from hydrogen (H) or deuterium (D).

[0062] In some embodiments of this application, compounds having the structure shown in the following formulas IV(a)-1 or IV(a)-2 are provided. [ka] [ka]

[0063] In some embodiments of this application, compounds having the structure shown in the following formula III(a) are provided. [ka] [Effects of the Invention]

[0064] This application provides antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof with improved pharmacokinetic properties. Improved pharmacokinetic properties result in reduced toxicity, enhanced safety and / or tolerability, improved efficacy, and ultimately, an improvement in the therapeutic time range / therapeutic concentration range of the target compound. [Brief explanation of the drawing]

[0065] [Figure 1] Figure 1 shows the killing rates of BT474 tumor cells with anti-HER2 bispecific antibodies (Expi HER2-1, Expi HER2-2, 23C2 HER2-1, and 23C2 HER2-2) and trastuzumab + pertuzumab combination therapy. [Figure 2] Figure 2 shows the killing rates of NCI-N87 tumor cells with anti-HER2 bispecific antibody, trastuzumab, T-DM1, and trastuzumab plus pertuzumab combination. [Figure 3] Figure 3 shows the killing rates of JIMT-1 tumor cells with anti-HER2 bispecific antibody, trastuzumab, T-DM1, and trastuzumab plus pertuzumab combination. [Figure 4]Figure 4 shows the results of inhibition of proliferation in BT474 tumor cells by anti-HER2 bispecific antibody, trastuzumab, and trastuzumab plus pertuzumab combination. [Figure 5] Figure 5 shows the effects of anti-HER2 bispecific antibody, PBS solvent control, and trastuzumab + pertuzumab (Per+Tra) combination therapy on changes in mouse tumor volume in N87 mouse xenotumor transplantation models of gastric cancer. [Figure 6] Figure 6 shows the effect on body weight change of mice in the efficacy of anti-HER2 bispecific antibody, PBS solvent control, and trastuzumab + pertuzumab combination against xenotumors in N87 mice with gastric cancer. [Figure 7] Figure 7 shows the structures of some exemplary anti-HER2 bispecific antibodies, where the dimer Fc is represented by one black chain (first Fc polypeptide) and another gray chain (second Fc polypeptide), one antigen-binding domain (first antigen-binding fragment) is shown with a diagonal fill, and the other antigen-binding domain (second antigen-binding fragment) is shown in white, the first antigen-binding fragment is scFv fused to the first Fc polypeptide, and the second antigen-binding fragment is Fab fused to the second Fc polypeptide. [Figure 8] Figure 8 shows the endocytosis activity of drug conjugates with different antibodies (monoclonal antibody-DDDXD and bispecific antibody-DDDXD) in NCI-N87 tumor cells. [Figure 9] Figure 9 shows the endocytosis activity of drug conjugates with different antibodies (monoclonal antibody-DDDXD and bispecific antibody-DDDXD) in SK-BR-3 tumor cells. [Modes for carrying out the invention]

[0066] "Interpretation and Definition" Unless otherwise specified, the following terms used in this application have the meanings set forth below. Certain terms, unless specifically defined, are understood in the ordinary sense of the art, rather than as undefined or ambiguous. For example, Singleton et al., Dictionary. See also: 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 ed.), People's Health Press, 2010. Where trade names are mentioned herein, they refer to the corresponding products or their active ingredients.

[0067] The term "substituted" refers to the substitution of one or more hydrogen atoms on a particular atom by a substituent, provided that the valence of that atom remains normal and the resulting compound is stable. If the substituent is oxygen substitution (=O), two hydrogen atoms are substituted. Oxygen substitution does not occur in aryl groups.

[0068] The terms “optional” or “optionally” indicate that the following matter or situation may occur but is not necessarily so, and such expression includes cases where the matter or situation occurs and cases where it does not. “Optionally substituted” of a group means that the group may or may not be substituted. For example, “optionally” substituted of an ethyl group with a halogen means that the ethyl group may not be substituted (CH2CH3), may be monosubstituted (e.g., CH2CH2F), may be polysubstituted (e.g., CHFCH2F, CH2CHF2, etc.), or may be completely substituted (CF2CF3). As those skilled in the art will understand, no substitution or substitutional form exists for a group containing one or more substituents that cannot exist and / or be synthesized spatially.

[0069] In this specification, "C m~n " means that the part in question has an integer number of carbon atoms within the specified range. For example, "C 1~6 " refers to a group with one carbon atom, two carbon atoms, and three carbon atoms. This refers to having elementary atoms, four carbon atoms, five carbon atoms, or six carbon atoms.

[0070] If a specific variable (e.g., R) appears one or more times in the composition or structure of a compound, it is defined independently each time it appears. Therefore, for example, if the target group is substituted by two Rs, each R is selected independently.

[0071] If the number of a particular connecting group is 0, for example, -(CH2)0-, it indicates that the connecting group is a covalent bond.

[0072] When a variable is chosen from a covalent bond, it indicates that the two groups connected by it are directly linked. For example, if L in ALZ represents a covalent bond, it indicates that the structure is actually AZ.

[0073] The term "halo" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0074] The term "hydroxyl group" refers to the -OH group.

[0075] The term "cyano group" refers to the -CN group.

[0076] The term "mercapto group" refers to the -SH group.

[0077] The term "amino group" refers to the -NH2 group.

[0078] The term "nitro group" refers to the -NO2 group.

[0079] The term "alkyl group" refers to C n H 2n+1 This refers to a hydrocarbon group whose general formula is C. The alkyl group may be straight-chain or branched. For example, the term "C 1~6 An "alkyl group" refers to an alkyl group containing 1 to 6 carbon atoms (for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, neopentyl group, hexyl group, 2-methylpentyl group, etc.). Similarly, the above definition applies to the alkyl (alkyl) portion of alkoxy groups, alkylamino groups, dialkylamino groups, alkylsulfonyl groups, and alkylthio groups.

[0080] The term "alkoxy group" refers to an -O-alkyl group.

[0081] The term "alkylamino group" refers to an -NH-alkyl group.

[0082] The term "dialkylamino group" refers to a -N (alkyl)2 group.

[0083] The term "alkylsulfonyl group" refers to an -SO2-alkyl group.

[0084] The term "alkylthio group" refers to an -S-alkyl group.

[0085] The term "alkenyl group" refers to an unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, either in a straight or branched chain, having at least one double bond. Non-exclusive examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, and 1,3-butadienyl groups.

[0086] The term "alkynyl group" refers to a linear or branched chain consisting of carbon atoms and hydrogen atoms, at least It also refers to an unsaturated aliphatic hydrocarbon group having one triple bond. Non-restrictive examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH3), 2-propynyl (-CH2-C≡CH), and 1,3-diaacetylenyl (-C≡CC≡CH).

[0087] The term "cycloalkyl group" refers to a carbon ring that is fully saturated and can exist as a monocycle, crosslinked ring, or spirocycle. Unless otherwise specified, such carbon rings are generally 3 to 10 membered rings. Non-exclusive examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, and adamantanyl groups.

[0088] The term "cycloalkenyl group" refers to a non-aromatic carbocyclic ring that is imperfectly saturated and can exist as a monocyclic, bridging, or spirocyclic ring. Unless otherwise specified, such carbocyclic rings are generally 5 to 8 membered rings. Non-exclusive examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, and cycloheptadienyl groups.

[0089] The term "heterocyclyl group" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not a fully unsaturated heteroaryl group) and can exist as a monocycle, bridging ring, or spirocycle. Unless otherwise specified, such heterocycles are generally 3-7 membered rings containing 1-3 (preferably 1 or 2) heteroatoms independently selected from sulfur, oxygen, and / or nitrogen. Non-exclusive examples of heterocyclyl groups include, but are not limited to, oxyranyl, tetrahydrofuryl, dihydrofuryl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, and tetrahydrothienyl groups.

[0090] The term "heterocycloalkyl group" refers to a fully saturated cyclic group that can exist as a monocycle, a bridging ring, or a spirocycle. Unless otherwise specified, the heterocycle is generally a 3-7 membered ring containing 1-3 (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, oxyranyl, thyranyl, and azilidinyl groups; non-limiting examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, and thietanyl groups; and examples of 5-membered heterocycloalkyl groups include tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, and tetrahydrofuryl groups. Examples of 6-membered heterocycloalkyl groups, including but not limited to hydropyrazolyl groups, include piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thiooxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl, and 1,4-dithianyl groups, and examples of 7-membered heterocycloalkyl groups include but not limited to azepanyl, oxepanyl, and thiepanyl groups. Monocyclic heterocycloalkyl groups having 5 or 6 ring atoms are preferred.

[0091] The term "aryl group" refers to an aromatic cyclic group that is either a monocyclic or polycyclic aromatic group with a conjugated π-electron system. For example, aryl groups have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Non-exclusive examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, and 1,2,3,4-tetrahydronaphthalene.

[0092] The term "heteroaryl group" refers to a group in which at least one ring atom is selected from N, O, and S, and the rest The ring atom of a heteroaryl group is carbon, and the group refers to a monocyclic or polycyclic ring system having at least one aromatic ring. A heteroaryl group preferably has one 4-8 membered ring, particularly a 5-8 membered ring, or multiple fused rings containing 6-14, particularly 6-10, ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuryl, benzothienyl, indolyl, and isoindolyl groups.

[0093] A "derivative" is a compound formed when an atom or group of atoms in a parent compound molecule is substituted by another atom or group of atoms, and is called a derivative of the parent compound.

[0094] Unless otherwise specified, any atom in the compound labeled and synthesized in this application may represent any stable isotope of that atom. Unless otherwise specified, a position in the structure is defined as H, i.e., hydrogen (H-1), so that position contains only naturally occurring isotopes. Similarly, unless otherwise specified, a position in the structure is defined as D, i.e., deuterium (H-2), so the isotopic amount at that position is at least 3340 times greater than the naturally occurring isotopic amount (0.015%) (i.e., containing at least 50.1% deuterium isotopes), and if one or more positions in the structure of the compound labeled and synthesized are defined as D, i.e., deuterium (H-2), the content of the compound shown in 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%. In this application, the deuterated ratio of the labeled and synthesized compound refers to the ratio of the isotopic content of the labeled and synthesized compound to the amount of naturally occurring isotopes. The deuterated ratio of each target deuterium atom in the labeled and synthesized compound may be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), at least 6600 times (99%), or at least 6633.3 times (99.5%). In this application, isotope species (isotopologues) refer to compounds that differ only in their isotopic composition in terms of chemical structure. The labeled and synthesized compounds in this application have the same chemical structure, differing only in the isotopic composition of the atoms in their molecules. Therefore, compounds containing deuterium at a specific position, which are labeled and synthesized according to this invention, also contain small amounts of hydrogen isotopes at that position. The amount of hydrogen isotopes at a particular position in the compounds labeled and synthesized according to this invention depends on many factors, including the purity of the deuterium isotope in the deuterating reagent (D2O, D2, NaBD4, LiAlD4, etc.) and the effectiveness of the synthesis method for introducing the deuterium isotope.However, the total amount of hydrogen isotopes at any given position is less than 49.9%. The total amount of hydrogen isotopes at any given position of the compounds labeled and synthesized in this application is less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1%, or 0.5%.

[0095] In this application, each atom not designated as deuterium exists in the abundance of its natural isotope.

[0096] The term “treatment” means administering the compounds or formulations described herein to prevent, improve or resolve a disease or one or more symptoms associated with said disease, and includes (i) preventing the onset of a disease or condition in a mammal, particularly when the mammal is susceptible to said condition but is not diagnosed with said condition; (ii) inhibiting the disease or condition, i.e., inhibiting its progression; and (iii) alleviating the disease or condition, i.e., reducing said disease or condition.

[0097] The term "therapeutic dose" means (i) the amount of a dose used to treat or prevent a specific disease, condition, or disorder. (ii) reducing, improving or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition or disorder as described herein. The “therapeutic dose” of the compound of the application will vary depending on the compound, the disease and its severity, the method of administration, and the age of the treated mammal, but can be determined by a person skilled in the art based on their knowledge and the content of this disclosure.

[0098] The term "pharmaceutically acceptable" is used for compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human or animal tissue, are non-toxic or irritating, are medically judged not to cause allergic reactions, other problems or complications, and have a reasonable benefit-to-risk ratio.

[0099] Examples of pharmaceutically acceptable salts include metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with organic acids, and salts formed with basic or acidic amino acids.

[0100] The term "solvate" refers to a substance formed when a compound associates with a solvent molecule.

[0101] The term "pharmaceutical composition" refers to a mixture consisting of one or more compounds of the present application or salts thereof and pharmaceutically acceptable additives. Pharmaceutical compositions are intended to facilitate the administration of the compounds of the present application to living organisms.

[0102] The term "pharmaceutically acceptable additive" refers to an additive that does not cause obvious irritation to living organisms and does not impair the biological activity and properties of the active compound. Suitable additives, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, and water, are well known to those skilled in the art.

[0103] The compounds and intermediates of this application may exist in different tautomer forms, and all such forms are included within the scope of this application. The term "tautomer" or "tautomer form" refers to structural isomers with different energies that can be interconverted by a low-energy barrier. For example, proton tautomers (also called prototropic tautomers) include interconversion by proton transfers, such as ketone-enol isomerization and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, in which a proton can be transferred between two nitrogen atoms on the ring. Valence tautomers include interconversion by rearrangement of several bonding electrons.

[0104] The term "antibody" is used in its broadest sense to include complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, multifunctional antibodies, and antibody fragments, as long as they possess the desired biological activity.

[0105] The term "humanized antibody" refers to an antibody that contains a CDR region derived from a non-human antibody, while the rest of the antibody molecule is derived from one or more human antibodies.

[0106] The term "mutant" refers to a peptide containing an amino acid sequence derived from the amino acid sequence of a peptide, such as by replacing one, two, or more amino acids with different amino acids from the original peptide, deleting one, two, or more wild-type amino acids, inserting one, two, or more amino acids that are not present in the wild type, and / or adding amino acids that are not present in the wild type to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of the wild type (hereinafter collectively referred to as "mutation"). In this application, "insertion" is included in "addition". It is.

[0107] The term "CDR (Complementarity-Determining Region)," also known as "hypervariable region," refers to each region of an antibody variable domain that forms a loop with a highly variable sequence and / or limited structure. Natural tetrabodies generally contain six CDRs: three located in the heavy chain variable region and three in the light chain variable region.

[0108] Regarding the term "variable region," an antibody structural unit is composed of two pairs of polypeptide chains, each pair having one heavy chain and one light chain. The variable region is a region limited to the N-terminal domain of each chain, consisting of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition.

[0109] The term "Fab" refers to a molecule that includes the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, as well as the variable domains VL (light chain variable region) and VH (heavy chain variable region) located in the light and heavy chains, respectively. The variable domains include the complementarity-determining region (CDR) which is involved in antigen binding.

[0110] The term "scFv" refers to an antibody comprising the VH and VL domains, where these domains are located on a single polypeptide chain. In some embodiments, the scFv further includes a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form the structure necessary for antigen binding.

[0111] The term "ECD" refers to the extracellular domain. HER receptors are receptor proteins belonging to the human epidermal growth factor receptor (HER) family, and include EGFR, HER2, HER3, and HER4 receptors. The HER2 receptor generally contains an extracellular domain capable of binding to HER ligands, a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxy-terminal signaling domain with several phosphorylated tyrosine residues. The extracellular domain of HER2 contains four domains: ECD1, ECD2, ECD3, and ECD4.

[0112] The term "antibody portion" refers to the antibody portion of an antibody-drug conjugate, which in some specific embodiments is linked to an intermediate linker portion by a specific functional group, and this antibody portion can specifically bind to an antigen.

[0113] The term "linker portion" refers to the part of an antibody-drug conjugate that connects the antibody portion and the cytotoxic drug portion. It can be cleavable or non-cleavable, and a cleavable linker is one that can be cleaved within the target cell to release the cytotoxic drug.

[0114] The term "cytotoxic drug portion" refers to the cytotoxic drug portion of an antibody-drug conjugate. In some specific embodiments, it is linked to the intermediate linker portion by a functional group, and within tumor cells, it releases cytotoxic drug molecules to exert an antitumor effect.

[0115] Trastuzumab is a recombinant humanized monoclonal antibody that selectively acts on the extracellular site of the human epidermal growth factor receptor-4 (HER4) and can be used to treat HER2-positive cancers. One example is a therapeutic monoclonal antibody product marketed under the trade name HERCEPTIN®.

[0116] Pertuzumab is a recombinant humanized monoclonal antibody that selectively acts on the extracellular site of the human epidermal growth factor receptor-2 (HER2) and can be used to treat HER2-positive cancers.

[0117] The term "HER2" refers to the second member of the EGFR family, which possesses tyrosine kinase activity. HER2 expression levels can be detected by immunohistochemistry. HER2-positive results refer to IHC3+, HER2-negative results refer to IHC1+ / 0, and IHC2+ results require further ISH detection.

[0118] The term "cancer" generally refers to a physiological disease in mammals characterized by uncontrolled cell proliferation.

[0119] The term "triple-negative breast cancer" refers to breast cancer in which estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 are all negatively expressed.

[0120] In this specification, unless otherwise specified, the terms "comprise, comprises, comprising" or equivalent terms (contain, contains, containing, include, includes, including) are open expressions meaning that, in addition to the elements, components, or steps listed, other elements, components, or steps not explicitly stated may also be included.

[0121] In this specification, unless otherwise specified, any numbers used herein to express amounts of components, measured values, or reaction conditions are understood to be modified in all contexts by the term “approximately.” When used in conjunction with a percentage, the term “approximately” may represent, for example, ±0.1%, preferably ±0.05%, and more preferably ±0.01%.

[0122] Unless explicitly indicated in the context, singular terms cover multiple referents, and vice versa. Similarly, unless explicitly indicated in the context, the term "or" includes the case of "and."

[0123] In this specification, the percentage of identity (degree of homology) between sequences can be determined by comparing two sequences using relevant computer programs (e.g., BLASTp or BLASTn with default settings) that are freely available from the World Wide Web (e.g., www.ncbi.nlm.nih.gov). [Examples]

[0124] Next, the present application will be further explained using examples, however, the scope of the present application is not limited to the examples. The reagents used in the present application are generally commercially available and can be used without purification.

[0125] The trastuzumab and pertuzumab used in the examples of this application are antibodies produced by a standard method, which involves first constructing a vector, transfecting eukaryotic cells, and then purifying and expressing them. The sequence of trastuzumab is referenced from WHO DRUG INFORMATION INN RL78, and the sequence of pertuzumab is referenced from the example portion of WO0100245. DS-8201 is the active ingredient of the commercially available product Enhertu manufactured by Daiichi Sankyo, and its structure is the same as that of trastuzumab-DXD produced in this application (see Example 15 for its structure).

[0126] Example 1: Construction, expression, and purification of anti-Her2 scFv-Fc and its variants. When constructing anti-Her2 scFv-Fc, the Fc portion uses human IgG1, and the anti-Her2 arm variable region sequence is a sequence based on Herceptin® monoclonal antibody, and Hercep is linked by the designed linker 1 (i.e., (GGGGS)3). The light and heavy chain variable regions of the tin(registered trademark) monoclonal antibody were ligated to form anti-Her2 scFv-Fc (SEQ ID NO: 1). Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0127] Furthermore, the following mutants were constructed by introducing point mutations into the anti-Her2 scFv-Fc sequence. Anti-Her2-scFv-VL-F53Y-Fc (SEQ ID NO: 3): Derived from wild-type anti-Her2 scFv-Fc, the VL region has an F53Y mutation. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASYLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0128] Anti-Her2-scFv-VL-F53A-Fc (SEQ ID NO: 5): Derived from wild-type anti-Her2 scFv-Fc, the VL region has the F53A mutation. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASALYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0129] Anti-Her2-scFv-VL-F53R-Fc (SEQ ID NO: 7): Wild-type anti-Her2 s It is derived from cFv-Fc, and the VL region has the F53R mutation. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASRLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0130] Anti-Her2-scFv-VH-K30E-Fc (SEQ ID NO: 9): Derived from wild-type anti-Her2 scFv-Fc, the VH region has a K30E mutation. Its amino acid sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFNIEDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0131] DNA sequences (SEQ ID NOs: 2, 4, 6, 8, 10) of anti-Her2 scFv-Fc and its variants were synthesized and cloned into pcDNA3.1 expression vectors. ExpiCHO cells (CHO-S, Thermo) were cotransfected with the anti-Her2 scFv-Fc or its variant expression vectors using the ExpiCHO® expression kit (Thermo Fisher, catalog number: A29133). The cells were placed in ExpiCHO expression medium and cultured in a 37°C × 8% CO2 humid atmosphere in an orbital shaker platform rotating at 130 rpm. The culture supernatant was collected and purified using Protein A magnetic beads (Genscript, catalog number: L00273). Protein concentration was measured using a UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific). [Table 5]

[0132] Example 2: Verification of aggregates of anti-Her2 scFv-Fc and its variants, and detection of binding to human Her2 antigen. The affinity of expressed and purified anti-Her2 scFv-Fc and its variants to the human Her2 protein was measured using Biacore T200(GE). The experimental procedure was as follows: A chip conjugated with anti-hIgG captured a certain amount of anti-Her2 scFv-Fc or its variant. Then, the antigen human HER2 protein (Sino Biological, catalog number: 10004-H08H) was flowed over the surface of the chip, and the reaction signal was detected in real time using a Biacore T200 to obtain binding and dissociation curves. The buffer used in the experiment was Biacore general-purpose buffer (137mM NaCl, 2.7mM The reagent consisted of KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, and 0.05% surfactant P20 (GE, catalog number: BR-1000-54), pH 7.4. When anti-hIgG (captured using a human antibody capture kit, GE, catalog number: 29-2346-00) bound to the surface of the CM5 chip, the reaction value was approximately 9000 RU. After capturing anti-Her2 scFv-Fc or its variants, the reaction value was approximately 200 RU. Subsequently, the signal values ​​of interactions between human HER2 protein at different concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) and anti-Her2 scFv-Fc or its variants were detected. The flow cell flow rate was 50 μL / min, the binding time was 240 seconds, and the dissociation time was 1400 seconds. Baseline stability was achieved after regeneration with 3M MgCl2(GE) for 60 seconds. Results were obtained by calculation using the affinity and kinetics 1:1 binding model of biacore evaluation software. See Table 2 for the affinity of anti-Her2 scFv-Fc or the mutant to the human Her2 protein antigen. Compared to anti-Her2 scFv-Fc (KD=0.77nM), the F53A mutation had a greater impact on the binding affinity of the mutant anti-Her2-scFv-VL-F53A-Fc (KD=1.26nM) to the human Her2 protein antigen. The binding affinity of the mutant anti-Her2-scFv-VL-F53Y-Fc (KD=0.8nM) to the human Her2 antigen was almost the same as that of anti-Her2 scFv-Fc (KD=0.77nM), while the KD of the mutant anti-Her2-scFv-VH-K30E-Fc to the human Her2 antigen was 0.54nM. It was found that the introduction of the F53Y and K30E mutations did not reduce the binding affinity to the human Her2 antigen.

[0133] Using a gel filtration chromatography column, each component of anti-Her2 scFv-Fc or its variants was separated, and the components were eluted in order of decreasing molecular weight. The chromatography column used was the ACQUITY UPLC Protein BEH SEC Column. Using a 200 Å gel filtration chromatography column with a 1.7 μm diameter and a 4.6 × 300 mm size. The column temperature was 25°C. The mobile phase was 50 mmol / L phosphate buffer - 200 mmol / L sodium chloride, with a pH of 7.0 (2.33 g of sodium dihydrogen phosphate dihydrate, 12.53 g of disodium hydrogen phosphate dodecahydrate, and 11.69 g of sodium chloride were weighed, approximately 800 mL of ultrapure water was added, and the mixture was stirred to dissolve completely. Then, ultrapure water was added to make a total volume of 1000 mL, and the mixture was homogeneously mixed before filtration through a 0.22 μm filtration membrane). The sample was diluted to 10 mg / mL in the mobile phase to prepare the test solution, and 2 μL was accurately weighed and injected into the liquid chromatograph (if the sample concentration was less than 10 mg / mL, the injection volume was adjusted to inject 20 μg of protein), and detection was performed at a wavelength of 280 nm. Isocratic elution was performed at a flow rate of 0.30 mL / min for 15 minutes. The data was processed, and the results were quantitatively analyzed using the area percentage method. The peak area percentages for aggregates, immunoglobulin monomers, and low molecular weight impurities were calculated, with aggregates present before the main peak, immunoglobulin monomers at the main peak, and low molecular weight impurities after the main peak. See Table 2 for the ratio of the main peak content to aggregates for anti-Her2 scFv-Fc or its variants. The variant anti-Her2-scFv-VL-F53Y-Fc and anti-Her2-scFv-VH-K30E-Fc clearly reduced aggregates. Aggregates were reduced from 6.31% in anti-Her2 scFv-Fc to 4.94% and 3.39%, respectively. [Table 6]

[0134] Example 3: Construction, expression, and purification of anti-Her2 bispecific antibody Modifications in the Knobs-into-holes (Ridgway, et al., 1996) Fc process generated a bispecific anti-Her2 antibody as human IgG1. The Fc region sequence of one of the heavy chains was designed with H435R and Y436F mutations (Jendeberg et al., 1997) to reduce the affinity of Fc for protein A, which helps remove homodimers formed during the assembly of the bispecific antibody in protein A affinity purification (Patent US5945311A). From Example 2, it was found that the anti-Her2-scFv-VL-F53Y-Fc mutation and the anti-Her2-scFv-VH-K30E-Fc mutation can significantly reduce anti-Her2-scFv aggregates without reducing affinity for the Her2 antigen. In this example, the antigen-binding domain of one anti-Her2 arm of the anti-Her2 bispecific antibody was in the form of scFv (VH-linker-VL structure), and the variable region sequence contained the mutation K30E (b-anti-Her2-scFv-VH-K30E-Fc, SEQ ID NO: 21) and the mutation F53Y (b-anti-Her2-scFv-VL-F53Y-Fc, SEQ ID NO: 23), or anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO: 11) containing both of these point mutations simultaneously. In this example, the antigen-binding domain of the other anti-Her2 arm of the anti-Her2 bispecific antibody is anti-Her2- This Fab form contains domain 2-HC-Fc (SEQ ID NO: 13) and anti-Her2-domain 2-LC (SEQ ID NO: 15). Furthermore, an anti-Her2 bispecific antibody was constructed as a control using scFv (VH-linker-VL structure or VL-linker-VH structure) that does not contain mutations. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5]

[0135] DNA sequences of anti-Her2 bispecific antibodies (SEQ ID NOs. 12, 14, and 16) were synthesized and cloned into pcDNA3.1 expression vectors. ExpiCHO cells were simultaneously transfected with the anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO. 11), anti-Her2-domain 2-HC-Fc (SEQ ID NO. 13), and anti-Her2-domain 2-LC (SEQ ID NO. 15) expression vectors using the CHOgro® High Yield Expression System (Product No.: MIR 6270) in a transfection ratio of 1:1:1.5. The transfection density was 6 × 10⁶. 6 The cell count was 1 / mL. The culture medium was CHOgro® expression medium (product number: MIR 6200, manufacturer: Mirus). Cells were continuously cultured for 10 days 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). The sample was named Expi Her2-2. Following this method, expression and purification were performed to produce other bispecific antibodies, Expi Her2-1 and Expi Her2-3, Expi Her2-4, and Expi Her2-5 were obtained.

[0136] Example 4: Production and validation of a fucose knockout bispecific antibody Knocking out the fucose expression-related gene FUT8 can improve the interaction between IgG1 and FcgRIIIa, thereby enhancing the ADCC effect of antibodies (Shields et al., 2002; Yamane-Ohnuki et al., 2004). In this example, fucose knockout anti-Her2 bispecific antibodies were produced using FUT8-knockout CHO-S cells (named CHO FUT8- / - cells). The DNA sequences of the anti-Her2 bispecific antibodies (SEQ ID NOs. 12, 14, and 16) were synthesized and cloned into pcDNA3.1 expression vectors. Expression vectors for anti-Her2-scFv-VH-K30E-VL-F53Y-Fc, anti-Her2-domain 2-HC-Fc, and anti-Her2-domain 2-LC were simultaneously transfected into FUT8-knockout CHO-S cells using the CHOgro® High Yield Expression System (product number: MIR 6270) in a transfection ratio of 1:1:1.5. The transfection density was 6 × 10⁶. 6 The cell count was 1 / mL. The culture medium was CHOgro® expression medium (product number: MIR 6200, manufacturer: Mirus). Cells were continuously cultured for 10 days 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). The sample was named 23C2 Her2-2. According to this method, in CHO FUT8- / - cells, Expi Her2-1, Expi Her2-3, Expi Her2-4, E The xpi Her2-5 sequence was expressed to obtain fucose knockout anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5.

[0137] Using the GlycoWorks RapiFluor-MS N-glycan kit (Waters, Milford, MA), samples of anti-Her2 bispecific antibody expressed by CHO FUT8- / - cells and CHO-S cells were processed to release N-glycans from the proteins. The N-glycans were then labeled, separated by a chromatography column, and analyzed with an FLR detector (Waters, Milford, MA) to obtain the structure and content of the N-glycans. See Table 4 for glycan content ratios. The fucose-free ratio for normal anti-Her2 bispecific antibody Expi HER2-2 was 21.85%, while the fucose-free ratio for anti-Her2 bispecific antibody 23C2 HER2-2 expressed by FUT8-knockout CHO-S cells was 99.40%. [Table 8-1] [Table 8-2]

[0138] Example 5: Verification of bispecific antibody aggregates This example relates to the validation of aggregates of the anti-HER2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, and 23C2 Her2-5.

[0139] Anti-HER2 bispecific antibodies were separated using a gel filtration chromatography column, and the aggregate content was verified. Each component was eluted in order of decreasing molecular weight. The chromatography column used was ACQUITY UPLC Protein BEH SEC Column. A 200 Å, 1.7 μm, 4.6 × 300 mm gel filtration chromatography column was used, with a column temperature of 25°C. The mobile phase was 50 mmol / L phosphate buffer - 200 mmol / L sodium chloride, with a pH of 7.0 (2.33 g of sodium dihydrogen phosphate dihydrate, 12.53 g of disodium hydrogen phosphate dodecahydrate, and 11.69 g of sodium chloride were weighed, approximately 800 mL of ultrapure water was added, and the mixture was stirred to dissolve completely. Ultrapure water was then added to make a total volume of 1000 mL, and the mixture was homogeneously mixed before filtration through a 0.22 μm filtration membrane). The sample was diluted to 10 mg / mL in the mobile phase to prepare the test solution. An accurate 2 μL was weighed and injected into a liquid chromatograph (if the sample concentration was less than 10 mg / mL, the injection volume was adjusted to inject 20 μg of protein), and detection was performed at a wavelength of 280 nm. Isocratic elution was performed at a flow rate of 0.30 mL / min for 15 minutes. The data were processed, and the results were quantitatively analyzed by the area percentage method. The peak area percentages for aggregates, immunoglobulin monomers, and low molecular weight impurities were calculated, respectively. Of these, aggregates were found before the main peak, immunoglobulin monomers were the main peak, and low molecular weight impurities were found after the main peak. [Table 9]

[0140] As is clear from the results, when a bispecific antibody is assembled from scFV, its bispecificity Although sex antibodies still produce large amounts of aggregates, introducing mutations reduces the aggregate content of bispecific antibodies. Compared to 23C2 Her2-1 without the mutation, the aggregate content of 23C2 Her2-2 was reduced to 8.51% (Table 5).

[0141] Example 6: Detection of antigen binding of anti-Her2 bispecific antibody The affinity of the HER2 protein of the test molecule was measured using Biacore T200(GE) for the expressed and purified anti-Her2 bispecific antibody, and the experimental procedure was as follows. A certain amount of anti-Her2 bispecific antibody was captured using a chip conjugated with anti-hIgG. Subsequently, human Her2 (Sino Biological, catalog number: 10004-H08H) was flowed over the surface of the chip, and the reaction signal was detected in real time using a Biacore T200 to obtain binding and dissociation curves. The buffer used in the experiment was Biacore general-purpose buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4, 0.05% surfactant P20, pH 7.4). When anti-hIgG (captured using a human antibody capture kit, GE, catalog number: 29-2346-00) bound to the surface of the CM5 chip and the reaction value reached approximately 9000 RU, the reaction value after capturing the anti-Her2 bispecific antibody was approximately 200 RU. Subsequently, the signal values ​​of the interaction between different concentrations of Her2 protein (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) and the anti-Her2 bispecific antibody were detected. The flow cell flow rate was 50 μL / min, the binding time was 240 seconds, and the dissociation time was 1400 seconds. After regeneration with 3 MgCl2 (GE) for 60 seconds, the baseline was stable.

[0142] The results were obtained by calculation using biacore evaluation software. Refer to Table 6 for the binding affinity of the anti-Her2 bispecific antibody 23C2 Her2-2 and the trastuzumab and pertuzumab control to the Her2 antigen. The KD of the anti-Her2 bispecific antibody 23C2 Her2-2 to the Her2 antigen was 6.11E-10M, the KD of the trastuzumab to the Her2 antigen was 1.22E-09M, and the KD of the pertuzumab to the Her2 antigen was 2.35E-09M. Her2-2 showed a higher affinity for the Her2 antigen than trastuzumab and pertuzumab. [Table 10]

[0143] Example 7: Detection of Antigen Binding of Anti-Her2 Bispecific Antibody According to the experimental procedure of Example 6, the affinity of the expressed and purified anti-Her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, 23C2 Her2-5 for HER2 protein can be measured by Biacore T200 (GE).

[0144] Refer to Table 7 for the measurement results of the affinity of the anti-Her2 bispecific antibody 23C2 Her2-1 for the antigen human Her2 protein. From the results of Table 6 and Table 7, it was found that introducing the F53Y and K30E mutations can improve the binding affinity of the anti-Her2 bispecific antibody for the antigen human Her2 protein.

Table 11

[0145] Example 8: Killing of Anti-Her2 Bispecific Antibody against Her2-Positive Target Cells BT474 With the provision of NK cells from human PBMC (peripheral blood mononuclear cells), the killing effect of the anti-Her2 bispecific antibody against target cells (BT474 Her2+++, provided by the Cell Bank of the Chinese Academy of Sciences Committee for the Preservation of Type Cultures) was examined, and the EC 50 value was used to evaluate the in vitro activity of the anti-Her2 bispecific antibody.

[0146] The details of the experimental procedure are as follows. For BT474 cells, the cell density was adjusted to 3×10 in 1640 experimental medium containing 2% FBS (fetal bovine serum). 5The cells were adjusted to the required concentration (cells / mL) and inoculated into 96-well cell culture plates (Eppendorf, product number: 0030730199) at 50 μL per well. Anti-Her2 bispecific antibodies were prepared at 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) using 1640 experimental medium, and 50 μL of each antibody concentration was added to the 96-well cell culture plates. Human PBMCs were cultured to a cell density of 1.5 × 10⁶ using 1640 experimental medium. 6 The volume was adjusted to cells / mL, resulting in 100 μL per well. The following groups were set up: 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). The effector cell-target cell ratio was 10:1. Forty-five minutes before detection, 20 μL / well of lysate (Promega, product number: G182A) was added to the target cell maximum release group and the lysate control group. After 45 minutes, cell lysis was detected using the CytoTox96® nonradioactive cytotoxicity assay (Promega, G1780). Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 ) × 100%.

[0147] Figure 1 shows the mortality rates of anti-Her2 bispecific antibodies against BT474 Her2+++ tumor cells. The ADCC-enhanced anti-Her2 bispecific antibodies (labeled 23C2 HER2-1 and 23C2 HER2-2 in Figure 1) were superior to trastuzumab + pertuzumab combination therapy and to anti-Her2 bispecific antibodies expressed from CHO-S (Expi HER2-1 and Expi HER2-2) in killing BT474 tumor cells. Among these, the trastuzumab + pertuzumab (1:1) combination therapy was superior to EC.50 The EC2 level was 8.627 ng / mL, and the EC2 level of Expi HER2-1 was 8.627 ng / mL. 50 It was 38.05 ng / mL, and Expi HER2-2 is EC 50 The EC level was 35.17 ng / mL, which was superior to that of Expi HER2-1. The EC level of 23C2 HER2-1 enhanced with ADCC was also higher. 50 The value was 4.728 ng / mL, and ADCC-enhanced 23C2 HER2-2 was EC 50 However, it was superior to 23C2 HER2-1 with enhanced ADCC at 3.658 ng / mL.

[0148] Example 9: Killing of Her2-positive target cells NCI-N87 by anti-Her2 bispecific antibody NK cells from human PBMCs (peripheral blood mononuclear cells) were used to develop anti-Her2 bispecific antibodies. The killing effect on target cells (NCI-N87 Her2++, provided by the Cell Bank of the Committee for the Preservation of Typical Cultures of the Chinese Academy of Sciences) was investigated, and EC 50 The in vitro activity of the anti-Her2 bispecific antibody was evaluated based on its size.

[0149] The details of the experimental procedure are as follows: NCI-N87 cells were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) to a cell density of 3 × 10⁻¹⁶ cells. 5 The cells were adjusted to the required cell / mL and inoculated into 96-well cell culture plates (Eppendorf, product number: 0030730199) at 50 μL per well. Anti-Her2 bispecific antibodies or control agents were prepared at different concentrations (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) using experimental media, and 50 μL of each concentration of antibody or control agent was added to the 96-well cell culture plates. Human PBMCs were then cultured to a cell density of 1.5 × 10⁶ using experimental media. 6The solution was adjusted to cells / mL, with 100 μL per well. The following groups were set up: treatment 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). The effector cell-target cell ratio was 10:1. 45 minutes prior to detection, 20 μL / well of lysate (Promega, product number: G182A) was added to the target cell maximum release group and the lysate control group. After 45 minutes, the CytoTox96® non-radioactive cytotoxicity detection kit (cytotox) was used. Cell lysis was detected using the 96 nonradioactive cytotoxicity assay (Promega, G1780). Trastuzumab, T-DM1 (trastuzumab-mytansin complex, trade name Kadcyla®), trastuzumab + pertuzumab (1:1), and Expi HER2-1 were used as control drugs. Dissolution rate (%)=(OD 投与群 -OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 -OD 標的細胞群 ) × 100%.

[0150] Figure 2 shows the killing rates of anti-Her2 bispecific antibodies against NCI-N87 tumor cells. The ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 was superior to trastuzumab + pertuzumab combination, trastuzumab, T-DM1, and Expi HER2-1 in killing NCI-N87 tumor cells. Among these, ADCC-enhanced 23C2 HER2-2 was superior to EC. 50 The value was 0.02447 nM, and the EC was treated with trastuzumab + pertuzumab combination therapy. 50 It is 0.08267nM, and the EC of Expi HER2-1 50 This is 0.1048nM, and the EC of T-DM1 50 The EC of trastuzumab is 0.07392 nM. 50 The value was 0.07468 nM.

[0151] Example 10: Killing of trastuzumab-resistant JIMT-1 cells by anti-Her2 bispecific antibody Using NK cells provided from human PBMCs (peripheral blood mononuclear cells), we investigated the killing effect of an anti-Her2 bispecific antibody on target cells (JIMT-1, provided by AddexBio, catalog number: C0006005) and EC 50 The in vitro activity of the anti-Her2 bispecific antibody was evaluated based on its size.

[0152] The details of the experimental procedure are as follows: JIMT-1 cells were cultured in 1640 experimental medium containing 2% FBS (fetal bovine serum) to a cell density of 3 × 10⁻¹⁶ cells. 5 The cells were adjusted to the required cell / mL and inoculated into 96-well cell culture plates (Eppendorf, product number: 0030730199) at 50 μL per well. Anti-Her2 bispecific antibodies or control agents were prepared at different concentrations (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) using experimental media, and 50 μL of each concentration of antibody or control agent was added to the 96-well cell culture plates. Human PBMCs were then cultured to a cell density of 1.5 × 10⁶ using experimental media. 6 The solution was adjusted to cells / mL, with 100 μL per well. Treatment group (target cells + effector cells + antibody or control drug), target cell group ( JIMT-1 Cells, effector cell population (human PBMC), target cell + effector cell population, blank control population (culture medium), lysate control population, and target cell maximal release population (target cell + lysate) were set up, and the effector cell-to-target cell ratio was 20:1. Forty-five minutes before detection, 20 μL / well of lysate (Promega, product number: G182A) was added to the target cell maximal release population and the lysate control population. After 45 minutes, the CytoTox96® nonradioactive cytotoxicity assay kit (Promega) was used. Cell lysis was detected using ega (G1780). Trastuzumab, T-DM1, trastuzumab + pertuzumab (1:1), and Expi HER2-1 were used as control drugs. Dissolution rate (%) = (OD 投与群 - OD 標的細胞+エフェクター細胞群 ) / (OD 標的細胞最大放出群 - OD 標的細胞群 ) × 100%.

[0153] Figure 3 shows the killing rate of the anti-Her2 bispecific antibody against JIMT-1 tumor cells. The killing of JIMT-1 tumor cells by the anti-Her2 bispecific antibody 23C2 HER2-2 with enhanced ADCC was superior to that of the combination of trastuzumab + pertuzumab, trastuzumab, T-DM1, and Expi HER2-1. Among them, the EC 50 of 23C2 HER2-2 with enhanced ADCC was 0.01006 nM, the EC 50 of the combination of trastuzumab + pertuzumab was 0.06727 nM, the EC 50 of Expi HER2-1 was 0.08066 nM, the EC 50 of T-DM1 was 0.08357 nM, and the EC 50 of trastuzumab was 0.07443 nM. Also, the anti-Her2 bispecific antibody 23C2 HER2-2 had a higher cell lysis rate.

[0154] Example 11: Growth inhibition of anti-Her2 bispecific antibody against BT474 Her2 +++ tumor cells 23C2 Her2-2, trastuzumab, and pertuzumab were diluted using DMEM / F12 medium (GIBCO, product number: 11330-032) containing 2% FBS (fetal bovine serum, GIBCO, product number: 10099-141) until the final concentration reached 3.2 μg / mL, and then serially diluted at a 1:1 ratio to obtain nine concentrations (1.6 μg / mL, 0.8 μg / mL, 0.4 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, 0.0125 μg / mL, 0.00625 μg / mL). For logarithmic growth phase BT474 Her2 +++ cells, the density was 1 × 10 5Adjusted to cells / mL and plated, adding 100 μL per well, and a cell-free blank well was set as a control. 50 μL of the serially diluted sample was added per well. It was cultured in a 37 °C × 5% CO₂ incubator for 5 days. The culture medium was discarded, 100 μL of CCK-8 (Dojindo Chemicals, product number: CK04) working solution was added per well, incubated for 4 - 5 hours for color development, and then placed in a microplate reader (Thermo, model number: VarioskanFlash), and the absorbance value at a wavelength of 450 nm was read and recorded at a reference wavelength of 630 nm. The growth inhibition rate of tumor cells was calculated.

[0155] As shown in the results of Figure 4, the growth inhibition rate of the anti-Her2 bispecific antibody 23C2 Her2-2 with enhanced ADCC against BT474 tumor cells was 78.38%, which was superior to the growth inhibition rate of trastuzumab (54.12%) and the growth inhibition rate of the combination of trastuzumab + pertuzumab (53.7%).

[0156] Example 12: Inhibitory effect of anti-Her2 bispecific antibody on xenograft tumors of NCI-N87 Her2++ gastric cancer nude mice In a mouse xenograft tumor transplantation model using NCI-N87 Her2++ gastric cancer cells (Cell Bank of the Chinese Academy of Sciences Committee for the Preservation of Type Cultures), the in vivo efficacy of the anti-Her2 bispecific antibody was evaluated. NCI-N87 Her2++ gastric cancer cells were prepared, and the concentration was 5 × 1 0 7 cells / mL, 0.1 mL per mouse. Under aseptic conditions, it was inoculated under the right rib of nude mice (provided by Changzhou ▼Ka▲wens Experimental Animal Co., Ltd., nude mice, 14 - 17 g, male, breeding environment: SPF). The diameter of the transplanted tumor was measured with a caliper for the tumors transplanted into the nude mice. When the tumor grew to 100 - 250 mm 3 the animals were divided into 5 groups. Group 1: Control Group 2: Expi Her2-1, 10 mg / kg Group 3: 23C2 Her2-2, 5 mg / kg Group 4: 23C2 Her2-2, 10 mg / kg Group 5: Per+Tra (trastuzumab + pertuzumab), 5 mg / kg + 5 mg / kg.

[0157] Each treatment group received intravenous injections at the prescribed dose, twice a week for three consecutive weeks (six doses). In group 5, the doses of the two drugs in the combination group were 5 mL / kg each, while the doses in all other groups were 10 mL / kg. Group 1 also received 10 mL / kg of PBS (Hyclone, product number: sh30256.01) intravenously. In the combination group, trastuzumab was administered at least 30 minutes after pertuzumab administration.

[0158] The antitumor effect of the test substance was dynamically observed using a tumor diameter measurement method. Tumor volume was measured 2-3 times per week, and the mouse's body weight was simultaneously measured and the data recorded. The general behavior of the mice was observed daily.

[0159] Detection indicators: Tumor volume (TV) is calculated using the formula TV = 1 / 2 × a × b 2 Here, a and b were the length and width of the tumor, respectively.

[0160] In this experiment, administration began on day d0 and was given a total of six times (d0, d3, d7, d10, d14, d17). There were no animal deaths by day d21 of the experiment. The average body weight of mice in each group tended to increase (Figure 6). The drug did not exhibit any apparent toxic effects.

[0161] Table 8 and Figure 5 show the effects of each test sample from Group 2 (10 mg / kg), Group 3 (5 mg / kg), Group 4 (10 mg / kg), and the Per+Tra combination group (5 mg / kg + 5 mg / kg) on ​​the volume of xenografted tumors in NCI-N87 gastric cancer nude mice up to day 21. The inhibition of xenografted tumors in NCI-N87 gastric cancer nude mice by the 23C2 Her2-2 group (10 mg / kg) was superior to that of Expi Her2-1 (10 mg / kg) and the Per+Tra combination group (5 mg / kg + 5 mg / kg). [Table 12]

[0162] Example 13: Synthesis of MC-GGFG-DXD [ka]

[0163] Step 1: Synthesis of A5 (({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl acetate) 20 g of SM5 (N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycine) was weighed and added to a 1 L three-necked flask. 300 mL of tetrahydrofuran and 100 mL of toluene were added, and the mixture was stirred to homogenize. 30 g of lead tetraacetate and 5.4 g of pyridine were added. The temperature was raised to 65°C and the reaction was allowed to proceed for 4 hours. After cooling to room temperature, the solid was removed by filtration. The organic phase was concentrated to dryness at 40°C. 300 mL of ethyl acetate and 300 mL of water were added to the concentrated organic phase. The mixture was stirred for 20 minutes. The ethyl acetate phase was separated. 100 mL of saturated sodium chloride solution was added to the ethyl acetate phase, and the mixture was stirred for 20 minutes. The ethyl acetate phase was separated and concentrated to dryness. Silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) was performed to obtain 13 g of A5. The yield was 63%. ESI-MS: m / z = 391.1 [M + Na] + .

[0164] Step 2: Synthesis of B5 ([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]benzyl acetate) 1.0 g of A5 was weighed and added to a 250 mL necked flask. 15 mL of DME (ethylene glycol dimethyl ether) and 0.897 g of benzyl glycolate were added, and the mixture was cooled to 0°C in an ice bath. A solution was prepared with 0.27 mL of water and 0.108 g of NaOH. The prepared NaOH solution was added to the reaction mixture. The reaction was allowed to proceed at 0°C for 1 hour, and 0.078 g of glacial acetic acid was added to the reaction mixture. 100 mL of water and 100 mL of ethyl acetate were added, and the mixture was stirred at room temperature for 20 minutes. The organic phase was separated and concentrated to dryness. Silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) was performed to obtain 0.68 g of B5. The yield was 53%. ESI-MS: m / z = 497.1 [M + Na] + .

[0165] Step 3: Synthesis of C5([({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid) 0.68 g of B5 was weighed and added to a 250 mL hydrogenation reaction bottle. 20 mL of ethanol, 10 mL of ethyl acetate, and 0.34 g of palladium / carbon water-wetted product (palladium content 10%) were added. Hydrogen gas was introduced using a hydrogen balloon. The reaction was allowed to proceed at room temperature for 1 hour. Palladium / carbon was removed by filtration through diatomaceous earth. The filtrate was concentrated to dryness at 40°C. 425 mg of C5 was obtained. The yield was 77%. ESI-MS: m / z = 407.1 [M + Na] + .

[0166] 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']indadino[1,2-b]quinoline-1-yl]amino}-2-oxoethoxy)methyl]glycinamide) Weigh 50 mg of SM4 (exatecan mesylate) and 50 mg of C5 and add them to a 100 mL single-neck flask. Add 2 mL of DMF (N,N-dimethylformamide), cool to 0 °C, and add 54 mg of HATU (O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate) and 30 mg of DIEA (N,N-diisopropylethylamine). Return to room temperature and react at room temperature for 3 hours. Add 100 mL of dichloromethane and 100 mL of water, stir for 20 minutes, separate the organic phase, and concentrate to dryness. Perform silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain 88 mg of D5. The yield was 84%. ESI-MS: m / z = 802.4 [M+H] + .

[0167] 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’]indazino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide) Weigh 78 mg of D5 and add it to a 100 mL single-neck 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 solution to dryness at 40 °C to obtain 55 mg of E5. The yield was 98%. ESI-MS: m / z = 580.3 [M+H] + .

[0168] Step 6: Synthesis of MC-GGFG-DXD 55 mg of E5 was weighed and added to a 100 mL necked flask. 2 mL of N,N-dimethylformamide and 48 mg of MC-GGF-OH (maleimidocaproylglycylglycylphenylalanine) were added, and the mixture was cooled to 0°C. 54 mg of HATU and 30 mg of DIEA were added. The mixture was allowed to return to room temperature. The mixture was reacted for 1 hour. 100 mL of ethyl acetate and 100 mL of water were added, and the mixture was stirred for 20 minutes. The organic phase was separated. The mixture was concentrated to dryness at 40°C. Silica gel column chromatography (ethyl acetate:methanol = 10:1) was performed to obtain 26 mg of MC-GGFG-DXD. The yield was 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(1 H,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),4.64 (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,d d,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).

[0169] Example 14: Synthesis of deuterated MC-GGFG-DXD (MC-GGFG-DDDXD), deuterated DXD (DDDXD), and DXD [ka]

[0170] Step 1: Synthesis of Intermediate A Under nitrogen protection, 80 g of ethyl diazo was added to a 3 L necked flask, and 800 mL of dichloromethane and 800 mL of 1% deuterium acetate solution (8 g of deuterium acetate dissolved in 800 mL of deuterium aqueous solution) were added. The reaction mixture was stirred at room temperature for 75 hours under the protection of light. The organic phase was collected by liquid-liquid extraction, and the aqueous phase was extracted twice with dichloromethane (200 mL x 2), and the organic phase was combined. The organic phase was washed with 200 mL of deuterium aqueous solution, the obtained organic phase was dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and the filtrate was concentrated to dryness under reduced pressure at 20°C to obtain 49.25 g of intermediate A. The yield was 66%. 1 H NMR (500MHz, CDCl3) δ 4.27 (q, J = 7.1 Hz, 1H), 1.31 (t, J = 7.1 Hz, 2H).

[0171] Step 2: Synthesis of Intermediate B Weigh 50g of N-fluorenylmethyloxycarbonyl-glycyl-glycine and 2 In a round-bottom flask, 750 mL of tetrahydrofuran and 150 mL of glacial acetic acid were added, and the mixture was stirred at 40°C for 20 minutes. 100 g of lead tetraacetate was added, and the temperature was raised to 80°C for an additional 3 hours. After cooling to room temperature, the mixture was filtered by suction, and the filter cake was washed with 250 mL of ethyl acetate. The filtrate was concentrated to dryness. 330 mL of dichloromethane and 670 mL of ethyl acetate were added to dissolve the mixture and obtain the organic phase. The organic phase was washed three times with 30% potassium bicarbonate aqueous solution (500 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure, 100 mL of dichloromethane was added to dissolve the mixture, and then 100 mL of n-hexane was added. The mixture was stirred at room temperature until a solid precipitate formed. Subsequently, 300 mL of a mixed solution of n-hexane and dichloromethane (n-hexane:dichloromethane = 1:1) was added, and the mixture was stirred overnight. The mixture was filtered, and the filter cake was dried in a vacuum oven at 40°C for 4 hours to obtain 37.3 g of intermediate B. The yield was 72%. LCMS(ESI)m / z:391.09[M+Na] + .

[0172] Step 3: Synthesis of intermediate C 78 g of intermediate B was weighed and added to a 3000 mL necked flask, then 800 mL of dichloromethane and 45 g of compound A were added. The 3000 mL necked flask was cooled to 0°C in an ice bath. 16 g of lithium tert-butoxide was dissolved in 400 mL of dichloromethane to prepare a lithium tert-butoxide solution. The lithium tert-butoxide solution was added to a 3000 mL round-bottom flask. The mixture was reacted at 0°C for 3 hours. The mixture was transferred to room temperature, 800 mL of water was added, and the mixture was stirred. The mixture was separated, and the organic phase was collected. The aqueous phase was extracted with 400 mL of dichloromethane. The organic phases were combined. The organic phases were washed once with 800 mL of saturated brine, dried over anhydrous sodium sulfate, filtered by suction, and the filtrate was concentrated under reduced pressure. 61 g of intermediate C was obtained by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1). The yield was 70%. LCMS(ESI)m / z:437.34[M+Na] + .

[0173] Step 4: Synthesis of Compound D 31.2 g of compound C was added to 270 mL of deuterium methanol and 70 mL of heavy water, and the mixture was stirred in an ice bath. Subsequently, 5.5 g of NaOH was added, and the mixture was transferred to room temperature and stirred overnight. 300 mL of ethyl acetate and 300 mL of water were added to the reaction mixture for extraction. 20 mL of glacial acetic acid was added to the aqueous layer to adjust the pH to 2-3. Once a solid precipitated, the mixture was filtered by suction to obtain 20.3 g of compound D. The yield was 69.8%. LC-MS (ESI) m / z: 409.08 [M + Na] + .

[0174] Step 5: Synthesis of Compound E 2.0 g of exatecan mesylate dihydrate and 1.63 g of compound D were weighed and added to a 100 mL round-bottom flask. 40 mL of N,N-dimethylformamide was added and the mixture was stirred. The mixture was cooled to 0°C. 2.0 g of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 1.82 g of N,N-diisopropylethylamine were added in that order. The mixture was reacted at 0°C for 3 hours. The reaction mixture was poured into 120 mL of ice water. The mixture was stirred for 1 hour and then filtered. The filter cake was dissolved in dichloromethane. Column chromatography (100 g of 100-200 mesh silica gel, dichloromethane:methanol = 30:1, 2 L) was performed to obtain 2.6 g of compound E. The yield was 92%. LCMS(ESI)m / z:804.84[M+H] + .

[0175] Step 6: Synthesis of Compound F 0.38 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene was weighed and added to a 100 mL round-bottom flask. 20 mL of tetrahydrofuran was added to the round-bottom flask and stirred. The mixture was cooled to 0°C. 2.0 g of compound E was weighed and a solution was prepared in 20 mL of tetrahydrofuran. The prepared solution of compound E was slowly poured into a 100 mL round-bottom flask. It was added to Sco. The reaction mixture was allowed to rise naturally to room temperature and reacted for 3 hours. It was filtered under nitrogen protection. 1.45 g of compound F was obtained. The yield was 98%. LC-MS (ESI) m / z: 582.39 [M+H] + .

[0176] Step 7: Synthesis of Compound H 1.00 g of compound F and 0.97 g of compound G were weighed and added to a 100 mL round-bottom flask, and 10 mL of N,N-dimethylformamide was added. The mixture was cooled to -20°C, and 0.34 g of 1-hydroxybenzotriazole and 0.49 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added. The mixture was reacted at -20°C for 3 hours. 20 mL of DCM and 20 mL of water were added to the reaction mixture, and the mixture was stirred for 30 minutes. After standing, the mixture was separated, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to dryness. Silica gel column chromatography (dichloromethane:methanol = 15:1) was performed to obtain 400 mg of compound H. The yield was 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). [ka] [ka]

[0177] Step 1: Synthesis of Intermediate I 0.85 g of compound A was added to 9 mL of deuterium methanol and 1 mL of heavy water, and the mixture was stirred in an ice bath. Subsequently, 0.32 g of sodium hydroxide was added, and the mixture was stirred overnight at room temperature. 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.

[0178] Step 2: Synthesis of compound J(DDDXD) 0.30 g of exatecan mesylate dihydrate and 0.056 g of intermediate I were added to 3 mL of N,N-dimethylformamide. The mixture was stirred in an ice bath, followed by the addition of 0.32 g of 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate and 0.22 g of N,N-diisopropylethylamine. The mixture was stirred at room temperature for 4 hours. The reaction mixture was subjected to liquid chromatography to obtain 0.18 g of compound J. The yield was 64.3%. LC-MS(ESI) m / z: 496.37[M+H] + . 1 H NMR(500MHz,DMSO-d6)8.39(d,J=8.9Hz,1H),7.70(d,J=10.9Hz,1H),7.29(s,1H),6.52(s,1H),5.58~5.54(m,1H),5.47(s,1H) ,5.40(s,2H),5.17~5.02(m,2H),3.24~3.03(m,2H),2.34(s,3H),2.25~2.09(m,2H),1.92~1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0179] Furthermore, DXD was manufactured according to the method described in Example 76 of the specification of Patent WO2014057687. [ka]

[0180] Example 15: Production of antibody-drug conjugates reagent: Solution A: PBS buffer with pH 7.4 Solution B: 10 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution Solution C: DMSO (dimethyl sulfoxide) Solution D: Histidine buffer (containing 0.89 mg / mL of L-histidine and 4.04 mg / mL of L-histidine hydrochloride monohydrate) Solution E: 700 mg / mL sucrose solution (prepared with Solution D) Solution F: 20 mg / mL polysorbate 80 (prepared with Solution D) Antibody: Trastuzumab, 23C2 Her2-2 Linker-payload (linker-cytotoxic drug portion): MC-GGFG-DXD, MC-GGFG-DDDXD [Table 13]

[0181] Experimental procedure: 1. Antibody replacement a. A 30 kD ultrafiltration centrifuge tube was thoroughly wetted with solution A. b. The antibody was replaced with solution A. c. An appropriate amount of solution A was added to adjust the antibody concentration to 5 mg / mL (23C2 Her2-2) and 7.5 mg / mL (trastuzumab).

[0182] 2. Antibody reduction a. The molar amount of the antibody was calculated and labeled N1. b. An appropriate amount of solution B was added to the antibody solution, and the molar amount of TCEP in the reaction system was set to N2. c. The mixture was wrapped in aluminum foil, placed in a rotary incubator, and shaken at a low speed (20 rpm) while being reacted in the dark at 37°C for 1 hour.

[0183] 3.Composite a. An appropriate amount of linker-payload was dissolved in DMSO, resulting in a final concentration of 10 mg / mL. b. DMSO was added to the antibody solution to adjust the antibody concentration to 5 wt%, and then an appropriate amount of linker-payload solution was added to adjust the molar concentration to N3. c. The mixture was wrapped in aluminum foil, placed in a rotary incubator, and shaken at a low speed (20 rpm) while being reacted in the dark at 20°C for 2 hours.

[0184] 4. Stop the compounding a. The ultrafiltration centrifuge tube was wetted with solution D. b. Replace the antibody with solution D, add appropriate amounts of solutions E and F, and adjust the concentrations of sucrose and polysorbate 80 to 90 mg / mL and 0.3 mg / mL, respectively, to -8 It was frozen and stored at 0°C.

[0185] Measurement of the DAR value (average number of drug conjugates per antibody molecule) of antibody-drug conjugates: DAR values ​​were measured using LC-MS. 50 μg of prepared ADC sample was mixed with 1 μL of glycosidase PNGase F (Ruian Bio, China) and incubated at 37°C for 20 hours. A high-resolution Xevo G2-XS mass spectrometer (Waters, USA) was used in the experiment. The sample concentration was adjusted to 5 μM, and mass spectrometry data in positive ion mode was collected using the direct injection method. The collected undenatured mass spectrometry data was processed using UNIFI 1.8.2.169 software (Waters, USA).

[0186] Measurement of protein concentration of antibody-drug conjugates: Protein concentration was detected using the Lowry method. Trastuzumab and 23C2 Her2-2 were used as standard substances. The absorbance OD at wavelength 650 was measured for the standard substances and the prepared ADC samples using a microplate reader. A standard curve was fitted, and the sample absorbance values ​​were substituted into the standard curve to calculate the protein concentration.

[0187] The following antibody-drug conjugates were manufactured and measured using the method described above. [ka] Trastuzumab-DXD, antibody concentration: 4.25 mg / mL, DAR: 7.6.

[0188] [ka] Trastuzumab-DDDXD, antibody concentration: 4.29 mg / mL, DAR: 7.7.

[0189] [ka] 23C2 Her2-2-DXD, antibody concentration: 4.35mg / mL, DAR: 5.7.

[0190] [ka] 23C2 Her2-2-DDDXD, antibody concentration: 4.16 mg / mL, DAR: 5.8.

[0191] Example 16: In vitro enzyme activity of deuterated DXD (DDDXD) 1. Preparation of reagents a. A 1% agarose electrophoresis gel was prepared. b. gelred staining solution was prepared and stored in a dark place. c. Preparation of topoisomerase I working solution: Prepared with ultrapure water and buffer solution.

[0192] 2. Sample preparation a. The compound (DDDXD) was reconstituted with DMSO and diluted, with the compound concentration ranging from 200 μM to a 10-fold dilution, creating five gradients.

[0193] 3. Reaction System a. Positive control: 15 μL ultrapure water + 2 μL 10× DNA topoisomerase I buffer + 2 μL 0.1% BSA + 1 μL pBR322 DNA b. Negative control: 14 μL ultrapure water + 2 μL 10× DNA topoisomerase I buffer + 2 μL 0.1% BSA + 1 μL pBR322 DNA + 1 μL topoisomerase I working solution. c. Sample group: 12 μL ultrapure water + 2 μL 10× DNA topoisomerase I buffer + 2 μL 0.1% BSA + 1 μL pBR322 DNA + 1 μL topoisomerase I working solution + 2 μL compound.

[0194] 4. Experimental Procedure a. The reaction system was placed in a water bath at 37°C for 30 minutes. b. 2 μL of loading buffer was added to the reaction system in each tube to stop the reaction. c. Agarose gel electrophoresis was performed at a voltage of 2-2.5 V / cm for 1.5 hours. d. The gels after electrophoresis were stained with GelRed in the dark for 1.5 hours and then imaged using a gel imager.

[0195] Example 17: Detection of antigen binding of antibody-drug conjugates The affinity of trastuzumab, 23C2 HER2-2, trastuzumab-DDDXD, and 23C2 HER2-2-DDDXD to the HER2 protein was measured according to the method of Example 6, and the results are shown in Table 10 below. [Table 14]

[0196] The results indicate that the anti-Her2 bispecific antibody has stronger antigen-binding activity than trastuzumab, and the anti-Her2 bispecific antibody ADC has stronger antigen-binding activity than trastuzumab ADC.

[0197] Example 18: Endocytosis experiment of antibody-drug conjugates Experimental method: For one bottle of logarithmic growth phase cells (NCI-N87 cells and SK-BR-3 cells), the cell density was set to 2.5 × 10⁻⁶. 6The solution was adjusted to the required number of cells / mL and added to a 96-well plate at 20 μL / well. The ADC samples trastuzumab-DDDXD and 23C2 HER2-2-DDDXD prepared in Example 15 were each pre-diluted to a concentration of 40 μg / mL, labeled as S1, and then subjected to 3-fold gradient dilution to obtain the corresponding samples S1-S9. DS-8201 and the control IgG1-DDDXD complex of IgG1 (non-HER2 target-specific IgG1, Yiqiao Shenzhou, product number: HG1K) and DDDXD were used as controls. The gradient-diluted sample solutions and labeled endocytosis reagent (Sartorius, 90564) were added to a cell culture plate at 20 μL per well and incubated at 37°C for 15 minutes. The 96-well cell culture plate was removed, and the two cell samples were added at 20 μL / well. The cell culture plate was then allowed to stand at 37°C and incubated for 2 hours. The 96-well cell culture plate was removed, placed in a flow cytometer, and the signal intensity was measured.

[0198] Refer to Figures 8 and 9 for the endocytosis experimental results on two HER2-positive cell types, NCI-N87 and SK-BR-3. The results show that endocytosis by anti-Her2 bispecific antibody ADCs is stronger than that of trastuzumab ADCs in both cases.

[0199] Example 19: Cellular activity of deuterated DXD and antibody-drug conjugates DXD and DDDXD were each pre-diluted to 140,000 ng / mL using culture medium, labeled as S1, and then subjected to a 5-fold gradient dilution to obtain corresponding reference samples S1-S9. The final drug concentrations ranged from 35,000 to 0.0896 ng / mL, totaling nine concentrations. Against HER2-positive tumor cells NCI-N87 in the logarithmic growth phase, the density was set to 1 × 10⁻⁶. 5The samples were plated at a concentration of cells / mL, 100 μL added per well, and a cell-free blank well was placed as a control. 50 μL of the two previously diluted samples were added per well. The samples were incubated in a 37°C × 5% CO2 incubator for 5 days. The culture medium was discarded, 100 μL of CCK-8 (Dojin Chemical, product number: CK04) working solution was added per well, and the plates were incubated for 4-5 hours to develop color. The plates were then placed on a microplate reader (manufacturer: Thermo, model number: VarioskanFlash), and the absorbance values ​​of the microplates at a wavelength of 450 nm were read and recorded at a reference wavelength of 630 nm. The inhibition rate of tumor cell proliferation was calculated.

[0200] For results regarding the NCI-N87 cell experiment, please refer to Table 11 below. [Table 15]

[0201] DDDXD exhibits stronger inhibitory activity against tumor cell proliferation than DXD.

[0202] The test antibody and the antibody-drug conjugate prepared in Example 15 were each pre-diluted to 20 μg / mL using culture medium, labeled as S1, and then subjected to a 5-fold gradient dilution to obtain corresponding samples S1-S9. The final drug concentrations ranged from 5000 to 0.0128 ng / mL, with a total of nine concentrations. Against HER2-positive tumor cells in the logarithmic growth phase (NCI-N87, BT474, and SK-BR-3), the densities were set to 2 × 10⁻⁶ for each sample. 4 The sample was adjusted to the cells / mL and plated, with 100 μL added per well. A cell-free blank well was then placed as a control. 50 μL of the gradient-diluted sample was added per well. 37°C × The cells were cultured in a 5% CO2 incubator. The culture medium was discarded, 100 μL of CTG detection solution (Promega, product number: G7572) was added per well, and the cells were incubated for 10 minutes to allow color development. The plates were then placed on a multifunction plate reader (manufacturer: Thermo, model number: VarioskanFlash) and the chemiluminescence values ​​were read. The inhibition rate of tumor cell proliferation was calculated.

[0203] For experimental results regarding NCI-N87 cells, please refer to Table 12 below. [Table 16]

[0204] For experimental results regarding BT474 cells, please refer to Table 13 below. [Table 17]

[0205] For experimental results regarding SK-BR-3 cells, please refer to Table 14 below. [Table 18]

[0206] The results above indicate that the cell-killing ability of anti-Her2 bispecific antibody ADCs is superior to that of trastuzumab ADCs.

[0207] Example 20: In vitro evaluation of liver microsome stability Each incubation system contained phosphate buffer (PBS, pH 7.4), liver microsomal proteins, substrate (acetonitrile solution of the test sample), and NADPH. The system was incubated in a 37°C water bath, and the reaction was stopped by adding an equal volume of cold acetonitrile at 0, 5, 15, 30, and 60 minutes after the reaction. A negative control was used, which was heat-inactivated by the corresponding species. The samples were incubated with liver microsomes. The remaining substrate content was detected by LC / MS / MS.

[0208] Example 21: In vivo pharmacokinetic experiment of antibody-drug conjugates The in vivo metabolic pathway and pharmacokinetic parameters of the antibody-drug conjugate of this invention were measured according 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.

[0209] Example 22: Inhibitory effect of antibody-drug conjugate on xenograft tumors in JIMT-1 Her2-positive breast cancer nude mice JIMT-1 breast cancer cells were prepared, and the concentration was set to 2 × 10⁻¹⁶. 7 The dose was 0.1 mL / mice. Under sterile conditions, the substance was inoculated into the right armpit of nude mice. After inoculation with the subcutaneous tumor graft, the tumor volume was 100-300 mm². 3 Once the animals reached a certain stage, they were randomly divided into three groups. The model group consisted of 6 mice in a solvent (containing 0.89 mg / mL L-histidine, 4.04 mg / mL L-histidine hydrochloride, 0.3 mg / mL polysorbate 80, and 90 mg / mL sucrose), 6 mice in the 23C2 Her2-2-DXD group (1.68 mg / kg, qw, iv), and 6 mice in the 23C2 Her2-2-DDDXD group (1.59 mg / kg, qw, iv). Tumor volume was measured 2-3 times a week, and the body weight of the mice was measured simultaneously, with the data recorded. The behavior of the animals was observed daily.

[0210] The formula for calculating relative weight (RWt) is as follows: RWC(%)=(W t / W t0 ) × 100% - 100% In the formula, W t0 This is the animal body weight per cage at the time of administration (i.e., d0), W t The value shown was the animal's body weight at the time of each measurement.

[0211] The formula for calculating tumor growth inhibition (TGI) is as follows: TGI(%) = (1 - TW / TW0) × 100% In the formula, TW was the tumor weight of the treatment group, and TW0 was the tumor weight of the model group.

[0212] For weight, please refer to Table 15 below. [Table 19]

[0213] For effectiveness, please refer to Table 16 below. [Table 20]

[0214] The results show that both 23C2 Her2-2-DXD and 23C2 Her2-2-DDDXD exhibit clear efficacy in the JIMT-1 mouse xenograft tumor model of human breast cancer cells, and that 23C2 Her2-2-DDDXD has a stronger antitumor effect than 23C2 Her2-2-DXD.

[0215] While the methods of the present application are described based on preferred embodiments as disclosed herein, those skilled in the art can modify or newly combine the products, elements, methods, and steps or the order of steps of the said methods without departing from the concept, spirit, and scope of the present application.

[0216] This specification explicitly incorporates, by reference to, all patents, patent applications, and other publications for explanatory and disclosure purposes. These publications are available because they were published prior to the filing date of this application. Statements regarding the disclosure dates or contents thereof of these documents are based on information available to the applicant and do not constitute an endorsement that the disclosure dates or contents thereof are accurate. Furthermore, the incorporation of these publications into this specification does not constitute an endorsement that such publications become common knowledge in the art in all applicable countries. All disclosures of the documents referred to herein are incorporated herein by reference and used to supplement the description herein by providing illustrative, procedural, and other details.

Claims

1. An antibody-drug conjugate having the general formula Ab-(L-U)n, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents the antibody moiety, L represents the linker moiety, U represents the cytotoxic drug moiety, and n is selected from an integer or decimal number between 1 and 10, wherein U is a camptothecin topoisomerase I inhibitor, and the L moiety and / or U moiety have deuterium modification. The aforementioned Ab comprises a monovalent first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells, and a monovalent second antigen-binding fragment that specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells. The first antigen-binding fragment is scFv and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 43, 28, and 29, and light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32. The second antigen-binding fragment is Fab, and the second antigen-binding fragment comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, wherein heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, and light chain CDR1, light chain CDR2, and light chain CDR3 each comprise the amino acid sequences shown in SEQ ID NOs. 48, 49, and 50, respectively, and is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

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

3. The antibody-drug conjugate comprises the structure shown in formula III or formula IV below, 【Chemistry 1】 Or, 【Chemistry 2】 In formula IV, R １ The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein is selected from hydrogen (H) or deuterium (D).

4. The antibody-drug conjugate comprises the structure shown in the following formula VI, 【Transformation 3】 In the formula, R １ , R ２ Each is independently selected from hydrogen (H) or deuterium (D). An antibody-drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or solvate thereof.

5. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein formula VI is the structure shown in VI-1, VI-2, VI-3, or VI-4 below. 【Chemistry 4】 【Transformation 5】 【Transformation 6】 Or, 【Transformation 7】

6. n is an integer or decimal number selected from 2 to 10, and is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5.

7. n is an integer or decimal number selected from 5 to 8, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 6.

8. The first antigen-binding fragment described above is i. The first antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each contain the amino acid sequences shown in SEQ ID NOs: 41 and 42, or ii. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from the first antigen-binding fragment having at least 80% identity with the amino acid sequences shown in SEQ ID NOs: 41 and 42, respectively.

9. The first antigen-binding fragment includes a heavy chain variable region and a light chain variable region, and the heavy chain The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 8, wherein the 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.

10. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 8 or 9, wherein the VH and VL of the first antigen-binding fragment are arranged in the order VH-linker-VL from the N-terminus to the C-terminus.

11. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 10, wherein the second antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region each comprising the amino acid sequences of SEQ ID NO: 37 and 38, respectively.

12. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 11, wherein the antibody portion Ab comprises an immunoglobulin functional domain operably linked to a first antigen-binding fragment and / or a second antigen-binding fragment, and the immunoglobulin functional domain comprises Fc.

13. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 12, wherein the Fc is a dimer Fc comprising a first Fc polypeptide and a second Fc polypeptide, the first antigen-binding fragment being operably linked to the first Fc polypeptide, and the second antigen-binding fragment being operably linked to the second Fc polypeptide.

14. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 13, wherein the antibody portion Ab is a bivalent bispecific antibody comprising a heavy chain of the sequence shown in SEQ ID NO: 11, a heavy chain of the sequence shown in SEQ ID NO: 13, and a light chain of the sequence shown in SEQ ID NO:

15.

15. It has the structure shown in the following formula VII, 【Transformation 8】 However, Ab represents the antibody portion and includes a first antigen-binding fragment that specifically binds to the ECD4 epitope of HER2 on HER2-expressing cells and a second antigen-binding fragment that specifically binds to the ECD2 epitope of HER2 on HER2-expressing cells, wherein the first antigen-binding fragment is scFv and consists of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light The heavy chain CDR1, heavy chain CDR2, and light chain CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 43, 28, and 29, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 30, 31, and 32, respectively, and the second antigen-binding fragment is Fab and contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, and the light chain CDR1, light chain CDR2, and light chain CDR3 each contain the amino acid sequences shown in SEQ ID NOs. 48, 49, and 50, respectively. n is selected from integers or decimals between 1 and 10. R １ , R ２ Each is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, each independently selected from hydrogen (H) or deuterium (D).

16. Formula VII is the structure shown in VII-1 below, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 15. 【Chemistry 9】

17. n is an integer or decimal number selected from 2 to 10, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 15 or 16.

18. n is an integer or decimal number between 5 and 8, wherein the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 17 is selected.

19. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 18, wherein the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 36, and the second antigen-binding fragment comprises 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 of SEQ ID NO: 37 and 38, respectively.

20. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of claims 15 to 19, wherein the antibody portion Ab is a bivalent bispecific antibody comprising a heavy chain of the sequence shown in SEQ ID NO: 11, a heavy chain of the sequence shown in SEQ ID NO: 13, and a light chain of the sequence shown in SEQ ID NO:

15.

21. A pharmaceutical product comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 20, for the prevention or treatment of cancer, wherein the cancer is HER2-positive cancer or cancer that is indicated as having HER2 expression as IHC2+ by immunohistochemistry.

22. The pharmaceutical product according to claim 21, wherein the cancer is breast cancer.

Images