Targeting TROP2 antibody-drug conjugate as well as preparation method and application thereof

Abstract

The invention provides an antibody-drug conjugate targeting TROP2, and a preparation method and application thereof. The TROP2-targeting antibody-drug conjugate shows very strong tumor specific cytotoxicity, and shows a stronger bystander effect in an in-vivo experiment, so that the TROP2-targeting antibody-drug conjugate can be used as a medical drug, especially a single drug or a combined drug for treating tumors, and used for neoadjuvant therapy of the tumors and the like.

Description

Antibody-drug conjugate targeting TROP2 and its preparation method and application Technical Field

[0001] The present application relates to an antibody-drug conjugate (ADC), and in particular to an antibody-drug conjugate targeting TROP2, and a preparation method and application thereof. Background Art

[0002] ADCs (antibody-drug conjugates) typically consist of three components: an antibody, a payload (e.g., a bioactive small molecule), and a linker. The payload is covalently coupled to the antibody via the linker. The antibody (e.g., a monoclonal antibody) can specifically recognize a target on the surface of tumor cells, guiding the ADC to the cancer cell surface and allowing it to enter the cancer cell through endocytosis. The ADC then releases the payload in the tumor microenvironment, specifically killing cancer cells without damaging normal tissue.

[0003] TROP2, a trophoblast cell surface antigen, also known as tumor-associated calcium signaling protein (TACSTD2), is overexpressed in a variety of human epithelial cancers, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer (Yezhe Cheng et al., Frontiers in Oncology, 2022 Dec 23;12:951589).

[0004] Preclinical and clinical studies have demonstrated the potential of anti-TROP2 antibody-drug conjugates (ADCs) (e.g., Trodelvy, an anti-human TROP2 antibody-SN-38 conjugate) for the treatment of cancer. Clinical results have shown promising therapeutic effects with Trodelvy in the treatment of refractory solid tumors. Trodelvy achieved an objective response rate (ORR) of 33% in patients with drug-resistant triple-negative breast cancer (TNBC). One of Trodelvy's key mechanisms of action is that the SN-38 (payload) is attached via a pH-sensitive linker, which cleaves and specifically releases the SN-38 in the acidic tumor microenvironment. However, Trodelvy's linker is not stable enough. The maleimide-mediated linker cleaves through thiol exchange under physiological conditions, resulting in a relatively short serum half-life of Trodelvy (approximately 1 day). Therefore, Trodelvy may have a relatively high off-target effect. There is a continued need for therapeutic antibody-drug conjugates targeting TROP2.

[0005] Summary of the Invention

[0006] Therefore, one aspect of the present application is to provide an antibody-drug conjugate having the following formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof

[0007] Wherein, D is represented by the following formula (II):

[0008] Among them, R 1 Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0009] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 6 and-SR 6 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 6 ; or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2;

[0010] R 6 is selected from H or C1-C4 alkyl; and

[0011] L1 is -L 1b -L 1a -, where L 1b Connect with Q', L 1a Connect with D,

[0012] Among them L 1a Does not exist or -(C1-C 10 alkylene)-;

[0013] L 1b Does not exist, *-(C1-C 10 Alkylene)-C(O)N(R 5 )-or*-(C1-C 10 Alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminus is covalently linked to Q'; and R 5 is H or C1-C6 alkyl,

[0014] Q' is -O- or -S-;

[0015] E is -CH2-NH-M-, wherein M is a peptide residue comprising 2 to 10 amino acids; wherein the peptide residue is optionally substituted by one or more (e.g., 2, 3 or 4) groups independently selected from C 1-6 and an alkyl and a polyol group; and wherein the N-terminus of said M is covalently attached to Z';

[0016] Z' is -C(=O)-L2-Y'-, wherein Z' is covalently linked to E via the -C(=O) moiety shown and to A via the Y' moiety;

[0017] L2 is selected from -(CH2) m -(O-CH2CH2) m1 -(CH2) m2 -、-(CH2) m -(O-CH2CH2) m1 -NHC(O)-(CH2) m -、-(CH2) m -(O-CH2CH2) m1 -C(O)NH-(CH2) m -, wherein m1 and m2 are independently selected from integers of 0-20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; m is selected from integers of 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8, wherein the left end of these groups is connected to -C(=O)- and the right end is connected to Y';

[0018] Y' is composed of The groups formed;

[0019] A represents an antibody or antibody fragment, preferably an antigen-binding fragment, targeting TROP2; and

[0020] p is the average drug to antibody ratio (ie, average DAR), which is a value between 1-15, such as 2-10, 2-8, 2-6, 2-5, 3-5, or 3.5-4.5.

[0021] In one embodiment, the present application provides an antibody-drug conjugate targeting TROP2, which has the structure of the following formula (I):

[0022] Wherein, D is represented by the following formula (II):

[0023] Among them, R 1 Selected from -H or C1-C4 alkyl; R 2 Selected from -H, -F, C1-C3 alkyl or C1-C3 haloalkyl; R 3Selected from -H, -F, -CN, -OCH3, -CH3 or -CF3;

[0024] L1 is independently unsubstituted or halogenated -(C1-C 10 alkylene)-;

[0025] Q' is -O- or -S-;

[0026] E is -CH2-NH-M, wherein M is a peptide comprising 2 to 10 amino acids; wherein optionally the amino acids are substituted with one or more polyols; and wherein the N-terminus of M is covalently attached to Z';

[0027] Z' is -C(=O)-L2-Y';

[0028] L2 is -(C1-C 10 alkylene)-;

[0029] Y'by form;

[0030] A represents an antibody or antibody fragment targeting TROP2; and

[0031] p is the mean drug to antibody ratio, with values ​​between 1-15.

[0032] The antibody or antibody fragment targeting TROP2 comprises a heavy chain and a light chain, wherein the heavy chain comprises a CDR1 having at least 80% sequence identity to the sequence shown by the amino acid sequence SEQ ID NO: 1, a CDR2 having at least 80% sequence identity to the sequence shown by the amino acid sequence SEQ ID NO: 2, and a CDR3 having at least 80% sequence identity to the sequence shown by the amino acid sequence SEQ ID NO: 3.

[0033] The light chain comprises a CDR1 having at least 80% sequence identity to the sequence shown by the amino acid sequence of SEQ ID NO: 4, a CDR2 having at least 80% sequence identity to the sequence shown by the amino acid sequence of SEQ ID NO: 5, and a CDR3 having at least 80% sequence identity to the sequence shown by the amino acid sequence of SEQ ID NO: 6.

[0034] In another aspect, the present application provides a composition comprising one or more antibody-drug conjugates according to the present application.

[0035] In another aspect, the present application provides use of the antibody-drug conjugate or the composition according to the present application in the preparation of a medicament for treating a cell proliferative disease or disorder or inhibiting abnormal cell growth.

[0036] In another aspect, the present application provides a method for preparing the antibody-drug conjugate according to the present application, the method comprising:

[0037] (i) preparing a linker-payload conjugate, wherein the linker-payload conjugate has the structure of the following formula (III):

[0038] ZE-Q'-L1-D Formula (III)

[0039] wherein the symbols are as defined herein; and

[0040] (ii) reacting the linker-load conjugate obtained in step (i) with an antibody or antibody fragment targeting TROP2. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 shows a schematic structural diagram of an ADC targeting TROP2.

[0042] FIG2A and FIG2B show the detection of the cytotoxic effect of ADC targeting TROP2 on the human hypopharyngeal carcinoma squamous cell line FaDu in vitro.

[0043] FIG3A and FIG3B show the detection of the cytotoxic effect of ADC targeting TROP2 on the human pancreatic cancer cell line BxPC-3 in vitro.

[0044] FIG4 shows the test results of the bystander effect of ADC targeting TROP2 in vitro.

[0045] Figures 5A and 5B show the anti-tumor effect of ADC targeting TROP2 in an in vivo human gastric cancer cell line NCI-N87 mouse xenograft tumor model.

[0046] Figure 6 shows the anti-tumor effect of ADC targeting TROP2 in an in vivo human lung cancer cell line HCC827 mouse transplant tumor model, A is the low-dose group, and B is the high-dose group.

[0047] FIG7 shows the anti-tumor effect of ADC targeting TROP2 in an in vivo human lung cancer cell line LK2 mouse xenograft tumor model.

[0048] FIG8 shows the anti-tumor effect of ADC targeting TROP2 in an in vivo human pancreatic cancer cell line BxPC-3 mouse xenograft tumor model.

[0049] FIG9 shows the anti-tumor effect of ADC targeting TROP2 in an in vivo human lung cancer cell line EBC-1 mouse xenograft tumor model.

[0050] FIG10A shows a schematic diagram of a method for detecting the stability of ADC targeting TROP2 in mice.

[0051] FIG10B shows the results of testing the stability of ADC targeting TROP2 in mice. DETAILED DESCRIPTION

[0052] Various embodiments of the TROP2-targeted ADC of the present application and methods of using the same will be described below with reference to the accompanying drawings. The following embodiments are given as exemplary embodiments of the embodiments of the present application and are not intended to limit the scope of the present application.

[0053] In one aspect, the present application provides an antibody-drug conjugate targeting TROP2 (also referred to as an "anti-TROP2 antibody-drug conjugate," "ADC targeting TROP2"). In the present application, the antibody-drug conjugate targeting TROP2 comprises an antibody targeting TROP2 and a drug connected by a linker.

[0054] Anti-TROP2 antibody-drug conjugates

[0055] In the present application, the present application provides an antibody-drug conjugate targeting TROP2, wherein the ADC targeting TROP2 is connected to an antibody targeting TROP2 and a camptothecin derivative via a peptide linker.

[0056] According to the antibody-drug conjugate targeting TROP2 of the present application, compared with the existing Ds-1062 (hTINA1-DXd) (Datopotamab deruxtecan, a novel TROP2-directed antibody-drug conjugate, demonstrates potent antitumor activity by efficient drug delivery to tumor cells, Daisuke Okajima et al., Mol Cancer Ther. 2021 Dec; 20 (12): 2329-2340), the ADC targeting TROP2 of the present application showed a stronger tumor suppression effect in multiple transplanted tumor models in vivo. In the in vitro experiments, it was found that the molecules of the present application had a better "bystander effect", which may be because the drugs of the ADC of the present application have better hydrophobic properties and can pass through the cell membrane more effectively. From the toxicity aspect, it was observed that the ADC targeting TROP2 of the present application did not cause severe weight loss in mice and the like, and showed good tolerance as a whole, which was not weaker than Ds-1062.

[0057] Compared with the existing Trodelvy (Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC), David M Goldenberg et al., Oncotarget. 2015 Sep 8; 6(26): 22496-512), the ADC targeting TROP2 of the present application has better stability (reference literature). The ADC targeting TROP2 of the present application has a stronger tumor inhibitory effect.

[0058] In addition, the small molecule drug of the present application has stronger hydrophobic properties and can better penetrate the cell membrane to enter the cell, thereby acting on the topoisomerase inside the cell and inhibiting cell growth. The ADC targeting TROP2 according to the present application showed a stronger "bystander effect" in in vivo experiments, thereby having a stronger tumor killing effect. Based on the above points, it can be inferred that the ADC targeting TROP2 according to the present application will show better tumor suppression effect in clinical practice.

[0059] In the present application, the ADC targeting TROP2 exhibits strong tumor-specific cytotoxicity and can therefore be used as a medical drug, especially as a single or combined drug for the treatment of tumors, as well as for neoadjuvant therapy of tumors.

[0060] In one aspect, the present application provides an antibody-drug conjugate having the following formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof

[0061] Wherein, D is represented by the following formula (II):

[0062] Among them, R 1 Selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl;

[0063] R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 6 and-SR 6 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 6 ; or R 2 and R 3 Together they form -O(CH2) nO- or -O(CF2) n O-, where n is 1 or 2;

[0064] R 6 is selected from H or C1-C4 alkyl; and

[0065] L1 is -L 1b -L 1a -, where L 1b Connect with Q', L 1a Connect with D,

[0066] Among them L 1a Does not exist or -(C1-C 10 alkylene)-;

[0067] L 1b Does not exist, *-(C1-C 10 Alkylene)-C(O)N(R 5 )-or*-(C1-C 10 Alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminus is covalently linked to Q'; and R 5 is H or C1-C6 alkyl,

[0068] Q' is -O- or -S-;

[0069] E is -CH2-NH-M-, wherein M is a peptide residue comprising 2 to 10 amino acids; wherein the peptide residue is optionally substituted by one or more (e.g., 2, 3 or 4) groups independently selected from C 1-6 and an alkyl and a polyol group; and wherein the N-terminus of said M is covalently attached to Z';

[0070] Z' is -C(=O)-L2-Y'-, wherein Z' is covalently linked to E via the -C(=O) moiety shown and to A via the Y' moiety;

[0071] L2 is selected from -(CH2) m -(O-CH2CH2) m1 -(CH2) m2 -、-(CH2) m -(O-CH2CH2) m1 -NHC(O)-(CH2) m -、-(CH2) m -(O-CH2CH2) m1 -C(O)NH-(CH2) m-, wherein m1 and m2 are independently selected from integers of 0-20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; m is selected from integers of 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8, wherein the left end of these groups is connected to -C(=O)- and the right end is connected to Y';

[0072] Y' is composed of The groups formed;

[0073] A represents an antibody or antibody fragment, preferably an antigen-binding fragment, targeting TROP2; and

[0074] p is the average drug to antibody ratio (ie, average DAR), which is a value between 1-15, such as 2-10, 2-8, 2-6, 2-5, 3-5, or 3.5-4.5.

[0075] It is understood that the N-terminus of M in the group E(-CH2-NH-M-) as described above is covalently attached to Z', and thus the "-CH2-" shown in E is covalently linked to Q'.

[0076] It should be understood that Y' is A group formed by A group formed by reaction with A (antibody or fragment thereof) which is covalently linked to A.

[0077] In some embodiments, M is a peptide residue having 2 to 10 amino acids, ie, a peptide residue consisting of 2 to 10 amino acids; which is optionally substituted as described above.

[0078] In one embodiment, the present application provides a TROP2-targeting antibody-drug conjugate having formula (I), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof:

[0079] Wherein, D is represented by the following formula (II):

[0080] Among them, R 1 Selected from -H or C1-C4 alkyl; R 2 Selected from -H, -F, C1-C3 alkyl or C1-C3 haloalkyl; R 3 Selected from -H, -F, -CN, -OCH3, -CH3 or -CF3;

[0081] L1 is independently unsubstituted or halogenated -(C1-C 10 alkylene)-;

[0082] Q' is -O- or -S-;

[0083] E is -CH2-NH-M, wherein M is a peptide comprising 2 to 10 amino acids; wherein optionally the amino acids are substituted with one or more polyols; and wherein the N-terminus of M is covalently attached to Z';

[0084] Z' is -C(=O)-L2-Y';

[0085] L2 is -(C1-C 10 alkylene)-;

[0086] Y'by form;

[0087] A represents an antibody or antibody fragment targeting TROP2; and

[0088] p is the mean drug to antibody ratio, with values ​​between 1-15.

[0089] In some embodiments, the present application provides a TROP2-targeting antibody-drug conjugate having formula (I) as defined above.

[0090] In some embodiments, Y' is Wherein, * represents the site covalently linked to the A.

[0091] In some embodiments, L2 is -(CH2) m -, m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5.

[0092] In an embodiment according to the present application, Z' is formed by:

[0093] In an embodiment, Z' is: or Wherein, * indicates the site of covalent attachment to A.

[0094] In some embodiments, the M is a peptide comprising 2, 3, or 4 amino acids, each amino acid of the M is an L amino acid, or at least one amino acid in the M is a D amino acid.

[0095] In some embodiments, M is a peptide having 2, 3 or 4 amino acids, ie, a peptide consisting of 2, 3 or 4 amino acids.

[0096] It will be understood that references to peptides and amino acid residues as part of a structure refer to either peptide residues or amino acid residues, as is well known to those skilled in the art.

[0097] In some embodiments, the amino acid is selected from, for example, glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine, and proline, and wherein the glutamine or glutamic acid is optionally substituted with a polyol group (e.g., 1 polyol group) and optionally substituted with C 1-6 Alkyl (e.g. 1 C 1-6 alkyl) substituted.

[0098] In some embodiments, the M comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol. In some embodiments, the M comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

[0099] In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol group and optionally substituted with a C 1-6 Alkyl substitution.

[0100] In some embodiments, the substituted glutamine or glutamic acid has the structure shown below:

[0101] where R 4 is H or C1-C6 alkyl;

[0102] Preferably

[0103] where R 4 It is H or C1-C6 alkyl.

[0104] In some embodiments, the substituted glutamine or glutamic acid has the structure shown below:

[0105] where R 4 is H or C1-C6 alkyl;

[0106] Preferably

[0107] where R 4 It is H or C1-C6 alkyl.

[0108] In some embodiments, M comprises an amino acid having the structure, Among them, R 4It is -H or C1-C6 alkyl.

[0109] In an embodiment, M comprises an amino acid having the structure

[0110] In some embodiments, M comprises or is a peptide residue selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Leu-Ala-*, -Ala-Leu-*, -Ala-Ala-*, -Phe-Gln-*, -Gln-Phe-*, -Leu-Gln-*, -Gln-Leu-*, -Val-Gln-*, -Phe-Ala-*, -Ala-Phe-*, -Gln-Val-*, -Ala-Ala-Ala-*, -Gly-Gly-Gly-*, -Ala-Val-Ala-*, -Gly-Val-Gly-*, -Ala-Val-Gly-*, -Gly-Phe-Gly-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val -Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Gln-*, -Ala-Val-Ala-Gly-*, -Gly-Phe-Gly-Gly-*, -Gly-Gly-Phe-Gly-*, -Gly-Phe-Gly-Gln-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala-*, -Ala-Val-Ala-Ala-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, -Gly-Phe-Gly-Gln-*, and -Gly-Leu-Phe-Gly-*, where * indicates the N-terminus of the peptide to which Z' is covalently attached.

[0111] In some embodiments, Gln is optionally substituted with 1 polyol group and optionally substituted with 1 C 1-6 Alkyl substituted.

[0112] In some embodiments, GIn is unsubstituted.

[0113] In some embodiments, Gln is substituted with a polyol group and / or with a C 1-6In some embodiments, M is selected from -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-*, -L-Ala-D-Val-L-Ala-*, -L-Ala-D-Ala-Gly-*, -L-Ala-D-Val-Gly-*, -L-Ala-L-Val-Gly-Gly-*, -L-Ala-L-Val-L-Gln-*, or -Gly-L-Phe-Gly-L-Gln-*, wherein * represents the N-terminus of the peptide to which Z' is covalently attached.

[0114] In some embodiments, -E- has one of the following structures, where * represents the N-terminus of the peptide to which Z' is covalently attached:

[0115] In some embodiments, Z'-E is formed from one of the following structures:

[0116] In some embodiments, Z'-E is selected from the following structures, where * indicates the point of attachment to A:

[0117] In some embodiments, R 1 、R 2 and R 3 At least one of them is not -H.

[0118] In some embodiments, R 1 is independently -H, methyl, ethyl or propyl.

[0119] In some embodiments, R 2 are independently -H, -F, methyl or ethyl; and R 3 is independently -H or -F.

[0120] In some embodiments, D is represented by the following structure:

[0121] In some embodiments, D is represented by the following structure: Among them, R 1 It is -H or C1-C3 alkyl.

[0122] In some embodiments, D is represented by the following structure:

[0123] In some embodiments, L1 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0124] In some embodiments, L1-Q' is -CH2CH2CH2CH2O-, -CH2CH2CH2O-, -CH2CH2O-, -CH2CH2CH2CH2S-, -CH2CH2CH2S-, or -CH2CH2S-.

[0125] In some embodiments, D-L1 is represented by the structure

[0126] In some embodiments, D-L1 is represented by the structure R 1 It is -H or C1-C3 alkyl.

[0127] In some embodiments, Q' is -O-.

[0128] In some embodiments, D-L1-Q'- has one of the following structures:

[0129] In some embodiments, D-L1-Q'-E-Z'- is formed by one of the following structures,

[0130] In some embodiments, is the following structure, wherein A is as defined herein, e.g., a monoclonal antibody, p is the average drug-to-antibody ratio (DAR), and p is a value between 1-15, 1-10, 2-8, 4-8 (including endpoints):

[0131] In some embodiments, the linker L is connected to the antibody (A) through a maleimide linker via an -S- group, wherein the -S- group is derived from the cysteine ​​side chain of A.

[0132] In one aspect, the present invention provides a compound represented by formula (I'), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof

[0133] wherein q is DAR and is an integer selected from 1-15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and

[0134] The other symbols such as A, Z', E, Q', L1 and D are as defined herein for formula (I).

[0135] The antibody or antibody fragment targeting TROP2 of the present application can be a mammalian-derived (e.g., human or mouse), humanized or chimeric antibody or antibody fragment targeting TROP2. Preferably, the antibody or antibody fragment targeting TROP2 is a monoclonal antibody recombinantly produced by cells genetically modified according to the techniques widely described in the prior art.

[0136] When A is an antibody targeting TROP2, it is preferably a human IgG, such as an IgG1, IgG2, IgG3 or IgG4 antibody.

[0137] According to the suggestion of the 3rd International Association for the Study of Lung Cancer (IASLC) Symposium on tumor and differentiation antigens, the RS7 antigen has been named EGP-1 (epithelial glycoprotein-1). At least one epitope associated with EGP-1 is also referred to as TROP2 in the literature. In a preferred embodiment, the epitope bound by the antibody or antibody fragment of the present application is the same as the epitope bound by the mouse RS7 antibody disclosed by Stein (Stein et al., Antibody Immunocon j. Radiopharm. 4: 703 (1991), which is incorporated by reference in its entirety) and other prior studies. Alternatively, the epitope bound by the antibody or fragment may be different from the epitope bound by the mouse RS7 antibody disclosed by Stein.

[0138] In some embodiments, the antibody or antibody fragment targeting TROP2 according to the present application comprises a heavy chain and a light chain, the heavy chain comprising a CDR1 having at least 80% sequence identity (preferably at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown by the amino acid sequence of SEQ ID NO: 1, a CDR2 having at least 80% sequence identity (preferably at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown by the amino acid sequence of SEQ ID NO: 2, and a CDR3 having at least 80% sequence identity (preferably at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity) to the sequence shown by the amino acid sequence of SEQ ID NO: 3.

[0139] The light chain comprises a CDR1 having at least 80% sequence identity (preferably at least 90% sequence identity, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity) to the sequence set forth in SEQ ID NO:4, a CDR2 having at least 80% sequence identity (preferably at least 90% sequence identity, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity) to the sequence set forth in SEQ ID NO:5, and a CDR3 having at least 80% sequence identity (preferably at least 90% sequence identity, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity) to the sequence set forth in SEQ ID NO:6.

[0140] For example, an antibody targeting TROP2 or a fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain includes a CDR1 comprising the amino acid sequence of GYTFTNYGM (SEQ ID NO: 1); a CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 2) and a CDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 3), and the light chain includes a CDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 4); a CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 5); and a CDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 6).

[0141] For example, a humanized antibody or fragment thereof targeting TROP2, wherein the complementarity determining region (CDR) of the light chain variable region of the humanized RS7 MAb includes a CDR1 containing the amino acid sequence KASQDVSIAVA; a CDR2 containing the amino acid sequence SASYRYT; and a CDR3 containing the amino acid sequence QQHYITPLT. In some embodiments of the humanized antibody or fragment thereof targeting TROP2 of the present application, wherein the CDR of the heavy chain variable region of the humanized RS7 MAb includes a CDR1 containing the amino acid sequence GYTFTNYGM; a CDR2 containing the amino acid sequence WINTYTGEPTYTDDFKG and a CDR3 containing the amino acid sequence GGFGSSYWYFDV. It is also preferred that the humanized antibody or fragment thereof further comprises FRs of the light chain and heavy chain constant regions of a human antibody.

[0142] In a specific embodiment, the antibody or fragment thereof targeting TROP2 comprises a variable domain of a light chain and a variable domain of a heavy chain, for example, the variable domain of the heavy chain includes a CDR1 comprising the amino acid sequence of GYTFTNYGM (SEQ ID NO: 1); a CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 2) and a CDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 3), and the variable domain of the light chain includes a CDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 4); a CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 5); and a CDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 6).

[0143] In a specific embodiment, the variable domain of the light chain of the antibody targeting TROP2 or its fragment has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:8, preferably at least 90% sequence identity, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity; and the variable domain of the heavy chain of the antibody targeting TROP2 or its fragment has at least 80% sequence identity, preferably at least 90% sequence identity, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with the amino acid sequence of SEQ ID NO:7.

[0144] In a specific embodiment, the antibody or fragment thereof targeting TROP2 comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:8; and the heavy chain variable domain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:7.

[0145] In a specific embodiment, the antibody targeting TROP2 or its fragment comprises a heavy chain and a light chain, wherein the light chain of the antibody targeting TROP2 or its fragment has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:10, preferably at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity; and the heavy chain of the antibody targeting TROP2 or its fragment has at least 80% sequence identity, preferably at least 90% sequence identity, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0146] In a specific embodiment, the antibody or fragment thereof targeting TROP2 comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 10. In a specific embodiment, A is the hRS7 antibody as shown in the Examples.

[0147] In a specific embodiment, the hRS7 antibody is an IgG1 humanized TROP2-targeting antibody having a light chain sequence of SEQ ID NO: 10 and a heavy chain sequence of SEQ ID NO: 9.

[0148] In a specific embodiment, the antibody targeting TROP2 is a full-length antibody.In a specific embodiment, the antibody targeting TROP2 or a fragment thereof comprises two heavy chains and two light chains, or consists of two heavy chains and two light chains.

[0149] In a specific embodiment, the antibody targeting TROP2 is a multispecific antibody such as a bispecific antibody.

[0150] In a specific embodiment, the antibody targeting TROP2 is a monoclonal antibody.

[0151] In a specific embodiment, the antibody targeting TROP2 is a chimeric antibody or a humanized antibody.

[0152] As an example, the relevant sequences of the hRS7 antibody are shown in Table 1 below.

[0153] Table 1

[0154] CDR definition rule: Kabat+Chothia

[0155] When p is equal to 1, the antibody-drug conjugate is generally referred to as "DAR1". When p is equal to 2, the antibody-drug conjugate is generally referred to as "DAR2". When p is equal to 3, the antibody-drug conjugate is generally referred to as "DAR3". When p is equal to 4, the antibody-drug conjugate is generally referred to as "DAR4", and so on.

[0156] Composition

[0157] The present application also relates to a composition comprising one or more antibody-drug conjugates of formula (I) as defined above, stereoisomers or pharmaceutically acceptable salts or solvates thereof. The composition may be a pharmaceutical composition comprising one or more antibody-drug conjugates of formula (I) as defined above, stereoisomers or pharmaceutically acceptable salts or solvates thereof, and a pharmaceutically acceptable carrier.

[0158] The compositions according to the present application have a particularly homogeneous character, which may result in better stability of the composition, better efficacy and / or reduced side effects compared to non-homogeneous compositions.

[0159] Advantageously, when A is an antibody or antibody fragment targeting TROP2 (e.g. hRS7), the composition according to the present application is characterized by the following features:

[0160] a) at least 50%, preferably at least 55%, at least 60%, at least 65% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) of the antibody-drug conjugates, stereoisomers, or pharmaceutically acceptable salts or solvates thereof of the composition have p equal to 1-15;

[0161] b) the average drug-to-antibody ratio (average DAR) is between 1 and 15, preferably between 1 and 10, between 2 and 8, or between 3 and 4. The average DAR is typically determined by HIC (hydrophobic interaction chromatography) or by native mass spectrometry;

[0162] c) at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) of the antibody-drug conjugate is in monomeric form. The monomer percentage is typically determined by SEC (size exclusion chromatography);

[0163] or d) a combination of two or three characteristics selected from a), b) and c).

[0164] Advantageously, when A is an antibody (e.g. hRS7), the composition according to the present application is characterized by the following features:

[0165] a) at least 50%, preferably at least 55%, at least 60%, at least 65% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the antibody-drug conjugates of the composition have a DAR of 4;

[0166] b) the average drug-to-antibody ratio (average DAR) is between 3.5 and 4.5; preferably between 3.8 and 4.2, between 3.9 and 4.1, between 3.9 and 4.0; for example, equal to 4.0 ± 0.2, 4.0 ± 0.1, for example, equal to 3.93 ± 0.01. The average DAR is usually determined by HIC (hydrophobic interaction chromatography) method, by native mass spectrometry or high-performance liquid chromatography (HPLC), such as RP-HPLC;

[0167] c) at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) of the antibody-drug conjugate is in monomeric form. The monomer percentage is typically determined by SEC (size exclusion chromatography);

[0168] or d) a combination of two or three characteristics selected from a), b) and c).

[0169] Drug combinations, medicine boxes

[0170] In one aspect, the present invention further provides a drug combination or drug combination product comprising an antibody-drug conjugate of formula (I) or (I'), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, and one or more other therapeutic agents. Other therapeutic agents that can be combined with the molecules of the present invention or administered in combination encompass various therapeutic agents for treating tumors, such as chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0171] In yet another aspect, the present disclosure relates to a kit for treating or preventing a TROP2-related disease, such as cancer, comprising an antibody-drug conjugate of formula (I) or (I'), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein, or a pharmaceutical composition comprising an antibody-drug conjugate of formula (I) or (I'), a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein, optionally a container and optionally a package insert or label indicating treatment.

[0172] Therapeutic uses, treatment methods

[0173] The present application also relates to the use of an antibody-drug conjugate according to formula (I) or (I') of the present application, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof, or a composition comprising an antibody-drug conjugate according to formula (I) or (I') of the present application, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof as a medicament, for example, for treating or preventing TROP2-related diseases, such as cell proliferative diseases or disorders, or an agent for inhibiting abnormal cell growth (e.g., cancer).

[0174] The cancer may be selected from adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, a CNS tumor, colon or colorectal cancer, diffuse intrinsic pontine glioma, endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, nasopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, kidney cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0175] The cancer may be selected from one or more of the following: lung cancer (non-small cell lung cancer, squamous cell carcinoma), pancreatic cancer, pharyngeal cancer, nasopharyngeal cancer, gastric cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer and prostate cancer.

[0176] In some embodiments, the cancer is a TROP2-positive cancer.

[0177] In some embodiments, a TROP2-positive cancer refers to abnormal expression or activity of TROP2 in a subject suffering from the cancer. In some embodiments, the subject (particularly an adult subject) has TROP2 expression. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) TROP2 (e.g., compared to a healthy subject). In some embodiments, the subject's biological sample (e.g., tumor cell or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) TROP2 (e.g., compared to a biological sample of a healthy subject (e.g., a corresponding tissue or cell in a healthy subject), or compared to TROP2 in an adjacent healthy tissue or cell of the subject).

[0178] In some embodiments, TROP2-positive cancer refers to a tumor cell expressing TROP2 in an individual with all cancers. In some embodiments, the tumor cells of the individual express TROP2, such as moderate or high expression of TROP2. In some embodiments, a TROP2-positive tumor refers to abnormal expression of TROP2 in tumor cells. In some embodiments, abnormal expression of TROP2 refers to high expression of TROP2 on tumor cells compared to control cells (e.g., healthy cells of corresponding tissues of healthy individuals, or healthy cells adjacent to tumor cells).

[0179] Antibody-drug conjugates according to the application, its stereoisomer or pharmaceutically acceptable salt or solvate or composition are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.As used herein, term " parenteral administration " represents the mode of administration (usually by injection) except that enteron and local administration are given, and includes but is not limited to intravascular administration, intravenous administration, intramuscular administration, intraarterial administration, intrathecal administration, intracapsular administration, intraorbital administration, tumor administration, intracardiac administration, intradermal administration and intraperitoneal administration, by injection, by perfusion through trachea and subcutaneous administration, intraarticular administration, capsule administration, subarachnoid administration, spinal column administration and sternum administration.Preferably given intravenously in the context of the application, such as by intravenous infusion.

[0180] The dosage of the antibody-drug conjugate given to a subject in need will vary according to many factors, including but not limited to route of administration, the type and severity of the condition being treated, the patient's condition, the patient's size, the patient's age, etc. Those skilled in the art can easily determine the desired dosage range based on these and other factors based on their knowledge in the field. Appropriate dosage can also be determined using animal models or clinical trials. For example, the typical dosage of an antibody-drug conjugate can be 0.3 mg / kg, 0.6 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 20 mg / kg, 30 mg / kg or more. Administration can be performed in a single dose, or more generally, in multiple doses. The administration regimen can include an initial loading dose, followed by a maintenance dose (e.g., weekly, every two weeks, every three weeks, monthly or longer). The duration of treatment can vary according to the condition being treated and the subject.

[0181] The antibody-drug conjugates or compositions according to the present application can be used as monotherapy or in combination with antibody drugs, other antibody-drug conjugates, small molecule drugs and / or other chemotherapeutic drugs, such as drugs with recognized therapeutic benefits in the condition under consideration. For example, these drugs may include paclitaxel, docetaxel, doxorubicin, cyclophosphamide, aromatase inhibitors (such as anastrozole), or antibodies for anti-cancer immunotherapy (such as anti-PD1 antibodies).

[0182] In order to prepare a pharmaceutical composition comprising an antibody or its fragment, the antibody or its fragment or ADC according to the application can be mixed with a pharmaceutically acceptable carrier or excipient. The composition can further include one or more other therapeutic agents suitable for treating or preventing cancer, such as breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumor, neurilemoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophagus, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hypertrophy (BPH), gynecomastia, rhabdomyosarcoma and endometriosis.

[0183] Formulations of the therapeutic agent can be prepared by mixing with physiologically acceptable carriers, excipients or stabilizers, for example in the form of a lyophilized powder, syrup, aqueous solution, lotion or suspension.

[0184] On the one hand, the present application also relates to a method for treating or preventing TROP2-related diseases such as cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to formula (I) or (I') of the present application, its stereoisomers or pharmaceutically acceptable salts or solvates, or a composition comprising an antibody-drug conjugate according to formula (I) or (I') of the present application, its stereoisomers or pharmaceutically acceptable salts or solvates.

[0185] The present application also relates to a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to formula (I) or (I') of the present application, or a composition comprising an antibody-drug conjugate according to formula (I) or (I') of the present application.

[0186] On the one hand, the present application also provides the use of an antibody-drug conjugate according to formula (I) or (I') of the present application, its stereoisomers or pharmaceutically acceptable salts or solvates, or a composition comprising an antibody-drug conjugate according to formula (I) or (I') of the present application, its stereoisomers or pharmaceutically acceptable salts or solvates in the preparation of a medicament for treating or preventing TROP2-related diseases such as cancer.

[0187] On the one hand, the present application also provides an antibody-drug conjugate according to formula (I) or (I') of the present application, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof, or a composition comprising an antibody-drug conjugate according to formula (I) or (I') of the present application, a stereoisomer or a pharmaceutically acceptable salt or solvate thereof, which is used as a drug, for example, for treating or preventing TROP2-related diseases such as cancer.

[0188] It should be understood that the TROP2-related disease or cancer is as defined herein.

[0189] Preparation method

[0190] The present description also relates to a method for preparing an antibody-drug conjugate of formula (I) or (I') as defined above, the method comprising:

[0191] (i) preparing a linker-payload conjugate, wherein the linker-payload conjugate has the structure of the following formula (III):

[0192] ZE-Q'-L1-D formula (III);

[0193] wherein E, Q', L1, and D are as defined for formula (I);

[0194] Z is as defined for Z', but where Y' is

[0195] as well as

[0196] (ii) reacting the linker-load conjugate obtained in step (i) with an antibody or antibody fragment targeting TROP2 as defined herein.

[0197] In some embodiments, prior to step (ii), the antibody or antibody fragment targeting TROP2 is treated with a thiol reducing agent (TCEP), and the treatment is performed using one or more of the following: (i) the concentration of the antibody or antibody fragment targeting TROP2 is 2 mg / mL-10 mg / mL, for example, the optimal concentration may be 5 mg / mL; (ii) the molar ratio of TCEP / mAb is 1.0 to 10.0, 2.0-6.0, for example, the optimal molar ratio is 2.0; (iii) the temperature is room temperature (20°C-37°C), and the optimal temperature is 25°C; (iv) the optimal pH value of the reaction is between 6.0 and 8.0; (v) the reaction time is 1-5h, 2-4h, for example, 2 hours.

[0198] In some embodiments, the antibody or antibody fragment targeting TROP2 is dissolved in PBS buffer.

[0199] In some embodiments, in step (ii), an excess of linker-load conjugate is reacted with the reduced antibody or antibody fragment targeting TROP2, and the reaction is carried out using one or more of the following: (i) the molar ratio of MB3 / mAb is 2.0-10.0, 6.0-10.00, for example, the optimal molar ratio is 6.0; (ii) the temperature is room temperature (20°C-37°C), and the optimal temperature is 25°C; (iii) the reaction time is 1-5h, 1-2h, for example, 2 hours.

[0200] In a preferred embodiment, after step (ii), the obtained crude ADC product is purified, for example, by spin desalting, ultrafiltration or dialysis purification, to obtain an ADC product.

[0201] This time, ADC products can be tested using RP-HPLC, LC-MS, and SEC HPLC to determine parameters such as DAR average and SEC purity.

[0202] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the examples.

[0203] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0204] definition

[0205] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following references are provided to provide many common definitions of the terms used in this application to those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed., 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0206] Unless expressly stated otherwise, the following terms used in this application shall have the following meanings as described herein.

[0207] The term "alkyl" refers to a group of the formula C n H 2n+1 The alkyl group may be straight chain or branched. For example, the term "C 1-6 The term "alkyl" refers to an alkyl group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio has the same definition as above.

[0208] The term "alkylene" as used as a molecule itself or as part of another molecule refers to a divalent free radical derived from an alkane. "Alkylene" is defined as alkyl as defined above, but is divalent, i.e., has two single bonds connecting to two other groups. For example, such groups include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-, all of which contain 10 or fewer carbon atoms, but the application is not limited thereto. The term "lower alkylene" refers to short alkylene groups generally having 6 or fewer carbon atoms, including, for example, "C 1-6 "Alkylene".

[0209] The term "alkylene" as used in this specification, examples and claims is intended to encompass both "unsubstituted alkylene" and "substituted alkylene". The latter refers to an alkylene group having a substituent replacing a hydrogen atom on one or more carbon atoms of a hydrocarbon. Unless otherwise expressly indicated, the substituent may include, for example, a halogen, a hydroxyl, a carbonyl (e.g., a carboxyl, an alkoxycarbonyl, a formyl or an acyl group), a thiocarbonyl (e.g., a thioester, a thioacetate or a thioformate), an alkoxyl, a phosphoryl, a phosphate group, a phosphonate group, a phosphinate group, an amino group, an amide group, an amidine group, an imine group, a cyano group, a nitro group, an azido group, a sulfhydryl group, an alkylthio group, a sulfate group, a sulfonic acid group, a sulfamoyl group, a sulfonamido group, a sulfonyl group, a heterocyclyl group, an aralkyl group, or an aromatic or heteroaromatic group. When suitably substituted, it will be understood by those skilled in the art that the substituted residue on the hydrocarbon chain itself may be substituted. For example, the substituent of the substituted alkylidene group can include replacement and unsubstituted amino, azido, imino, amido, phosphoryl (including phosphonate / ester and phosphinate / ester), sulfonyl (including sulfate / ester, sulfonamido, sulfamoyl and sulfonate / ester) and silyl, and can also include ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester) ,-CF ,-CN and its equivalent.Exemplary substituted alkyl is as described below.Cycloalkylidene can further be replaced by the alkyl substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl ,-CF ,-CN and its equivalent replace.

[0210] The term "alkenyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and including at least one double bond and no triple bonds. Alkenyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0211] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and at least one triple bond. Alkynyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

[0212] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0213] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. Halogenated alkyl groups may preferably be monohalogenated alkyl, dihalogenated alkyl, or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine, or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halo groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhalogenated alkyl groups refer to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0214] The term "haloalkenyl" refers to an alkenyl group, as defined herein, substituted with one or more halo groups, as defined herein. The term "haloalkynyl" refers to an alkynyl group, as defined herein, substituted with one or more halo groups, as defined herein. The meaning of "halo" as defined for "haloalkyl" applies to both "haloalkenyl" and "haloalkynyl."

[0215] The term "polyol group" refers to an alkyl group as defined above containing a plurality (e.g. 2-10, e.g. 3, 4, 5, 6, 7 or 8) of hydroxyl groups, which optionally contains 1 or more (e.g. 2, 3 or 4) other groups (e.g. amino, carbonyl). Non-limiting examples of "polyol groups" include, for example Among them, the chiral center without the indicated stereo configuration can be R or S configuration, preferably

[0216] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. Conventional amino acids are referred to herein in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Also, in this disclosure, amino acids are generally referred to by single-letter and three-letter abbreviations known in the art. For example, the amino acid can be selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro) and glutamine (Gln).

[0217] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, when a group or structure is "optionally substituted," the group or structure may be substituted or unsubstituted.

[0218] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention and is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed between the ADC conjugates of the present invention and an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed with organic bases containing an N group.

[0219] The term "solvate" refers to an association formed between one or more solvent molecules and the ADC antibody-drug conjugate of the present invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, and the like.

[0220] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0221] In the present application, the antibody-drug conjugates (Formula (I) or (II)) of the present invention may have one or more chiral (asymmetric) centers. The present disclosure encompasses all stereoisomeric forms of the antibody-drug conjugates. Asymmetric centers may have (R) or (S) configurations independently of one another. When the bond to a chiral carbon is described as a straight line in the structural formula of the present disclosure, or when the compound name is described without the (R) or (S) chiral designation of the chiral carbon, it is understood that the (R) and (S) configurations of each such chiral carbon and therefore each enantiomer or diastereomer and mixtures thereof are included in the formula or name. The production of a specific stereoisomer or mixture thereof can be identified in the examples in which such stereoisomers or mixtures are obtained, but this in no way limits all stereoisomers and mixtures thereof to being included within the scope of the present disclosure. When the bond of a chiral carbon is depicted as a solid line or a dashed line in a structural formula of the present disclosure, or when a compound name is depicted with a (R) or (S) chiral designation of a chiral carbon, it should be understood that the compound represented by the structural formula or name has a definite stereoconfiguration at that chiral carbon position and is to be distinguished from other stereoisomers, enantiomers, diastereomers, or mixtures thereof.

[0222] The term "drug:antibody ratio," "drug to antibody ratio," or "DAR" refers to, for an antibody drug conjugate molecule, the ratio of the drug moiety (D) conjugated to the A (antibody) moiety described herein to the A moiety, i.e., the number of drug moieties (D) attached per antibody (A). It will be understood that the DAR can be determined by q in Formula II. For example, the DAR can be an integer from 1 to 16, such as an integer from 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. For antibody drug conjugate products, the average DAR is usually used for characterization, i.e., the total ratio of the drug portion (D) coupled to the portion A described herein to the portion A in the product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), which is referred to herein as the average DAR or measured DAR. The average DAR can be an integer or a decimal. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 3-5, 3.5-4.5, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or any range having two of these values ​​as endpoints. It should be understood that when referring to an average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules comprising ADC molecules with the same and / or different DARs.

[0223] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0224] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0225] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0226] The term "drug" refers to an organic compound capable of modulating biological processes, particularly altering or preventing pathological processes.

[0227] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate biological processes, particularly alter or prevent pathological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, more preferably less than 500 kD. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0228] The "Drug (D)" portion of the antibody-drug conjugates of the present invention may be substituted with isotopes, including but not limited to deuterium and tritium, referred to as deuterated compounds and tritiated compounds, respectively. For example, when deuterium is substituted, a carbon-deuterium bond replaces a carbon-hydrogen bond. Since the former is more stable than the latter, this substitution can directly affect the absorption, distribution, metabolism, and excretion properties of certain drugs, thereby improving the drug's efficacy, safety, and tolerability. Therefore, the "Drug (D)" portion of the antibody-drug conjugates of the present application may include compounds substituted with deuterium or tritium.

[0229] "Deuterium-substituted" means that a hydrogen in the molecule is replaced by deuterium, eg, 1 or more hydrogens, eg, 1-10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) are replaced by deuterium.

[0230] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. If there are special explanations and descriptions, they should be understood in accordance with such special explanations and descriptions. In this article, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of or consisting of the stated elements, integers, or steps is also encompassed. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to encompass an antibody variable domain consisting of the specific sequence.

[0231] The compounds and intermediates of the present application can also exist in different tautomeric forms, and all such forms are included in the scope of the present application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. The specific example of a proton tautomer is the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions by reorganization of some bonding electrons.

[0232] As used herein, the term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, and is a protein or polypeptide that exhibits binding specificity for a specific antigen. Specific examples of antibodies may include full-length antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are heterotetramers composed of two heavy chains of approximately 50-70 kDa each (heavy chains are referred to as H chains) and two light chains of approximately 25 kDa each (light chains are referred to as L chains) linked together by intrachain and interchain disulfide bonds. Each chain consists of a variable region or variable domain at the N-terminal position, referred to as VL for the light chain and VH for the heavy chain, and a constant region at the C-terminal position of each chain, the constant region consisting of a single domain called CL in the light chain and three or four domains called CH1, CH2, CH3, and CH4 in the heavy chain.

[0233] Based on the amino acid sequence of the constant region of its heavy chains, antibodies can be divided into five major classes: IgA, IgD, IgE, IgG, and IgM. These antibody classes are further divided into subclasses based on the size of the hinge region, the location of interchain disulfide bonds, and molecular weight, such as IgG1, IgG2a, IgG2b, and IgG3. Based on the amino acid composition and arrangement of the constant region of its light chains, light chains can be divided into two types: kappa and lambda. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known in the art.

[0234] The term "chimeric antibody" is understood to mean an antibody in which the sequences of the light chain variable region and the heavy chain variable region and the sequences of the light chain constant region and the heavy chain constant region belong to different species. For the purposes of this application, the sequences of the heavy chain variable region and the light chain variable region are preferentially derived from murine sources, while the sequences of the heavy chain constant region and the light chain constant region belong to non-murine species. In this regard, for the constant region, all types of non-murine mammals can be used, particularly humans, monkeys, porcines, bovines, equines, felines, canines, and even birds, and this list is not exhaustive. Preferably, the chimeric antibody according to the present application comprises sequences of heavy and light chain constant regions of human origin and sequences of heavy and light chain variable regions of murine origin.

[0235] The term "humanized antibody" is understood to refer to antibodies in which the sequences of all or some of the regions involved in antigen recognition (hypervariable regions or CDRs: complementarity determining regions) and (sometimes) certain amino acids in the FR regions (framework regions) are non-human, while the sequences of the constant and variable regions not involved in antigen recognition are human.

[0236] The term "human antibody" is understood to refer to antibodies that comprise exclusively human sequences for both the variable and constant regions of the light chain and the variable and constant regions of the heavy chain.

[0237] The term "antibody fragment" is understood to refer to any portion of an immunoglobulin obtained by enzymatic digestion or by biological production, such as Fab, Fab', F(ab')2, Fab'-SH, scFv, or scFv-Fc. In some embodiments, the antibody is an antigen-binding fragment, and an "antigen-binding fragment" of an antibody refers to an amino acid fragment of an antibody molecule that participates in antigen-specific binding.

[0238] Enzymatic digestion of immunoglobulins by papain produces two identical fragments, which are called Fab (antigen binding fragment) fragments and Fc fragments (crystallizable fragments). Enzymatic digestion of immunoglobulins by pepsin produces Fc fragments and F(ab')2 fragments that are split into multiple peptides. F(ab')2 consists of two Fab' fragments connected by interchain disulfide bonds. The Fab portion consists of the variable region and the CH1 and CL domains. The Fab' fragment consists of the Fab region and the hinge region. Fab'-SH refers to a Fab' fragment in which the cysteine ​​residues in the hinge region carry a free thiol group. scFv (single-chain variable fragment) is a fragment obtained from protein engineering that consists only of the VH and VL variable domains. The structure is stabilized by a short, flexible peptide arm called a linker located between the two domains. The scFv fragment can be connected to the Fc fragment to form scFv-Fc.

[0239] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0240] The term "pharmaceutically acceptable carrier" can be used in the context of an excipient, diluent, or adjuvant. The carrier can be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, normal saline, buffers such as PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The carrier can include a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, or a combination thereof.

[0241] "Subject," "individual," or "patient" includes vertebrates. Particularly preferred examples of animals include mammals, e.g., cattle, dogs, horses, cats, sheep, pigs, and primates (including humans and non-human primates), especially humans. Preferably, it refers to an animal in which the molecules of the present application can achieve therapeutic or preventive effects.

[0242] The term "treatment" means administering the compound or formulation described herein to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0243] (i) preventing a disease or disease state from occurring in a mammal, particularly where such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state;

[0244] (ii) inhibiting the disease or disease state, i.e., curbing its development;

[0245] (iii) ameliorating the disease or condition, i.e., causing regression of the disease or condition.

[0246] The term "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats or prevents a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and this disclosure.

[0247] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody drug conjugate of the present invention, its stereoisomers or pharmaceutically acceptable salts or solvates, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in separate entities, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient. In some embodiments, the antibody drug conjugate of the present invention, its stereoisomers or pharmaceutically acceptable salts or solvates and other therapeutic agents used in the drug combination are administered at a level no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0248] The term "combination therapy" or "drug combination" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat the diseases described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes the co-administration of each active ingredient in a plurality of or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0249] The terms "about or approximately" refer to an amount, level, value, amount, frequency, percentage, dimension, size, portion, weight or length that varies by 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference amount, level, value, amount, frequency, percentage, dimension, size, portion, weight or length.

[0250] All patents, patent applications and other identified publications are expressly incorporated herein by reference for the purposes of description and disclosure. Any citation of these publications herein does not constitute an admission that the publications become part of the common general knowledge in the art.

[0251] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

[0252] Example

[0253] hTINA1 (the antibody used in Ds-1062) is a positive control antibody, and its sequence is derived from WHO Drug Information Vol. 34, No. 3, 2020. IgG is a negative control antibody from Equitech-bio, SLH56-0001.

[0254] Recombinant human TROP2 protein: 10428-H08H (Sino Biological); Rhesus trifoliate TROP2 protein: 90893-C08H (Sino Biological); mouse TROP2: 50922-M08H (Sino Biological); rat TROP2: 86246-R08H (Sino Biological).

[0255] Trodelvy is a positive control ADC, with catalog number information (GTIN (01) 0888818320019; LOT (10S22F010; Serial (21) FWDED608C5Y2). The antibody portion is hRS7, and the linker-payload is CL2A-SN38 (EMEA / H / C / 005182 / 0000). The structure is as follows:

[0256] Example 1 Preparation of Antibodies

[0257] Antibody preparation:

[0258] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was checked one day before transfection (viability should be greater than 95%) and adjusted to 3 × 10 6 The cell density was adjusted to 3×10 cells / mL on the day of transfection. 6 cells / mL;

[0259] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, where the light and heavy chain plasmids are mixed at a ratio of 1:1, add PEIMax (Polysciences Inc. Cat# 24765-1) at a DNA:PEI mass ratio of 1:3, mix well, and incubate at room temperature for 20 minutes. Then, gently pour the mixture into the Expi293F cell suspension while shaking. Incubate the cells on a shaker at 8% CO2, 36.5°C, and 120 rpm.

[0260] After 16-18 h of incubation, the cell suspension was supplemented with 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution (final concentration of 5 g / L), and valproic acid sodium salt (Merk, Cat# P4543-100G) (final concentration of 2.2 mM). The suspension was gently mixed and incubated for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat# 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0261] Purify target protein by affinity chromatography:

[0262] A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1×PBS to remove non-specific binding proteins. The packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. The pH was adjusted to 6.0 with 2 M Tris, filtered and sterilized, and after passing the purity test, the antibody coupling for ADC was carried out.

[0263] Example 2: Anti-TROP-2 Antibody Binding Activity Detection

[0264] 1. Determination of the affinity (KD) of antibodies hRS7 and hTINA1 prepared in Example 1 for binding to TROP2 of different species

[0265] Affinity measurements were performed using biofilm thin-layer interferometry (BLI). Half an hour before the start of the experiment, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20) based on the number of samples. Antibodies hRS7 and human TROP2 (10428-H41H, Sino Biological), rhesus TROP2 (90893-C08H, Sino Biological), mouse TROP2 (50922-M08H, Sino Biological), and rat TROP2 (86246-R08H, Sino Biological) were diluted to 100 nM. SD buffer, antibody solution, human TROP2, rhesus TROP2, mouse TROP2, and rat TROP2 were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using the Fortebio Octet Red96e, with the sensor position selected based on the sample placement. Instrument parameters were set as follows: Run steps: 120s baseline equilibration, 100s addition of immobilized antibody, 120s baseline equilibration, 100s antigen binding, and 120s dissociation, at a speed of 1000 rpm and a temperature of 30°C. After the experiment was completed, KD values ​​were analyzed using ForteBio Octet analysis software, and the results are shown in Table 2. Kinetic parameters from the affinity experiment indicate that hRS7 binds to human TROP2 and rhesus monkey TROP2 with affinities of 4.52nM and 7.75nM, respectively, and does not bind to mouse or rat TROP2 (data not shown).

[0266] Table 2: Affinity of antibodies hRS7 and hTINA1 for TROP2 from different species

[0267] 2. Testing the binding of TROP2 antibodies to human TROP2 protein at the cellular level

[0268] Add 10% FBS (SH30406.05, HYCLONE) to EMEM (30-2003, ATCC) medium and mix well to prepare FaDu cell (ATCC) growth medium. Adjust the FaDu cell concentration to 1x10 6 cells / mL, 1x10 5The cells were plated into 96-well plates (3799, Corning), centrifuged and washed twice with FACS buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). The antibody was diluted with FACS buffer, starting with 200nM, 3-fold gradient dilution, a total of 9 gradients, and the last concentration was 0nM. The diluted antibody was resuspended in the plate with cells and stained at 4°C for 30 minutes. After staining, the cells were washed twice with FACS buffer and continued to be stained with fluorescently labeled secondary antibodies (410708, Biolegend) at room temperature for 20 minutes. After staining, the cells were washed twice with FACS buffer and detected by FACS machine (CytoFLEX, BECKMAN). The data were fitted by GraphPad Prism and the IC of the drug was calculated. 50 (Table 3) The expressed hRS7 antibody was found to have similar affinity to the control antibody hTINA1 (the antibody used in Ds-1062).

[0269] Table 3: Test results of the ability of TROP2 antibodies to bind to cell surface TROP2 protein

[0270] Example 3: Preparation of Compound MB3 and Linker-Dxd

[0271] MB3 was prepared according to the method disclosed in Example 4 of WO2021 / 173773.

[0272] According to the method disclosed in Example 14 of WO2015 / 098099A1, Linker-Dxd shown in the following formula was prepared.

[0273] Example 4: Preparation of antibody-drug conjugates targeting TROP2

[0274] 4.1 Preparation of hRS7-MB3 according to the present application

[0275] (a) Dissolve the hRS7 antibody in PBS buffer (pH 7.4);

[0276] (b) adding a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water), and incubating the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal concentration of hRS7 is 5 mg / mL, (ii) the optimal molar ratio of TCEP / mAb is 2.0, (iii) the optimal reaction temperature is 25°C, and (iv) the optimal reaction pH is between 6.0 and 8.0;

[0277] (c) adding an excess of MB3 (dissolved in DMSO) to react with the antibody reduced in step (a), and the reaction mixture is placed at room temperature for 2 hours, wherein: (i) the optimal molar ratio of MB3 / mAb is 6.0, and (ii) the optimal reaction temperature is 25° C., thereby obtaining a crude ADC product;

[0278] (d) the obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product;

[0279] (e) ADC products were tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR and SEC purity.

[0280] Among them, the DAR average value was 4.3 and the SEC purity was 98.42.

[0281] 4.2 Preparation of hRS7-DXd and hTINA1-DXd

[0282] (a) Antibody hRS7 or antibody hTINA1 was dissolved in PBS buffer (pH 7.4);

[0283] (b) adding a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water), and incubating the reaction mixture at 0-10°C for 12 hours, wherein: (i) the optimal concentration of hTINA1 is 5 mg / mL, (ii) the optimal molar ratio of TCEP / mAb is 5.0, (iii) the optimal reaction temperature is 0-4°C, and (iv) the optimal reaction pH is between 6.0 and 8.0;

[0284] (c) adding an excess amount of Linker-DXd prepared in Example 3 (dissolved in DMSO) to react with the antibody reduced in step (a), and placing the reaction mixture at 0-4° C. for 1-2 hours, wherein: (i) the optimal molar ratio of DXd / mAb is 8, and (ii) the optimal reaction temperature is 0-4° C., thereby obtaining a crude ADC product;

[0285] (d) the obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product;

[0286] (e) ADC products were tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR and SEC purity.

[0287] The average DAR values ​​of hRS7-DXd and hTINA1-DXd were 4.4 and 4.3, respectively, and their SEC purities were 99.29 and 97.59, respectively.

[0288] Example 5 Preparation of IgG-drug conjugates

[0289] 5.1 Preparation of IgG-MB3

[0290] (a) Antibody IgG1 (isotype control) was dissolved in PBS buffer (pH 7.4);

[0291] (b) adding a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water), and incubating the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal concentration of IgG1 (isotype control) is 10 mg / mL, (ii) the optimal molar ratio of TCEP / mAb is 2.0, (iii) the optimal reaction temperature is 25°C, and (iv) the optimal reaction pH is between 6.0 and 8.0;

[0292] (c) adding an excess of MB3 (dissolved in DMSO) to react with the antibody reduced in step (a), and the reaction mixture was placed at room temperature for 2 hours, wherein: (i) the optimal molar ratio of MB3 / mAb was 6.0, and (ii) the optimal reaction temperature was 25° C., thereby obtaining a crude ADC product.

[0293] (d) the obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product;

[0294] (e) ADC products were tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR and SEC purity.

[0295] Among them, the DAR average value is 3.6 and the SEC purity is 99.34.

[0296] 5.2 Preparation of IgG-DXd

[0297] (a) Antibody IgG1 (isotype control) dissolved in PBS buffer;

[0298] (b) adding a reducing agent solution (TCEP, Aldrich, Catalog Number 646547, dissolved in water), and incubating the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal concentration of IgG1 (isotype control) is 10 mg / mL, (ii) the optimal molar ratio of TCEP / mAb is 2.0, (iii) the optimal reaction temperature is 25°C, and (iv) the optimal reaction pH is between 6.0 and 8.0;

[0299] (c) adding an excess amount of Linker-DXd prepared in Example 3 (dissolved in DMSO) to react with the antibody reduced in step (a), and the reaction mixture was kept at room temperature for 2 hours, wherein: (i) the optimal molar ratio of DXd / mAb was 6.0, and (ii) the optimal reaction temperature was 25° C., thereby obtaining a crude ADC product;

[0300] (d) the obtained crude ADC product is purified by spin desalting, ultrafiltration or dialysis to obtain the final ADC product;

[0301] (e) ADC products were tested using RP-HPLC, LC-MS, and SEC HPLC to determine the average DAR and SEC purity.

[0302] Among them, the DAR average value was 4.2 and the SEC purity was 98.69.

[0303] Effect Example 1: In vitro efficacy verification of TROP2-targeted antibody-drug conjugate (ADC targeting TROP2)

[0304] 1. In vitro cytotoxicity testing of ADCs targeting TROP2

[0305] Prepare BxPC3 cell growth medium (ATCC) by adding 10% FBS (SH30406.05, HYCLONE) to RPMI 1640 (22400-071, Gibco) and mixing thoroughly. Adjust the BxPC3 or FaDu cell concentration to 50,000 cells / mL using growth medium. Plate 50 μL per well (2,500 cells / well) into a 96-well white-bottom plate (167008, NUNC) and incubate overnight at 37°C. Dilute the antibody in growth medium using a 5-fold dilution series starting at 200 nM (2x) and continuing through nine steps, ending at 0 nM. Add 50 μL of the diluted antibody-drug conjugate or control to each well for a total volume of 100 μL. Seal the edges of the plate with 200 μL of PBS (pH 7.4) and incubate at 37°C for 6 days. Remove the cultured 96-well plate and CTL (DD1101-02, Vazyme) and equilibrate to room temperature. Add an equal volume of CTL (100 μL) to each well. Shake on a plate shaker for 5 minutes to fully lyse the cell aggregates. Allow the plate to stand at room temperature for 10 minutes to stabilize the luminescence signal. Analyze the plate on a microplate reader (spectra MAX i3x, Molecular Devices), read the A450 value, and calculate cell viability.

[0306] Table 4: In vitro cytotoxicity experiments of ADC targeting TROP2

[0307] The cell proliferation inhibition rate after 6 days of culture was calculated using the following formula.

[0308] Cell proliferation inhibition rate (%) = a / b*100, where a: the average value of the wells with experimental samples added after 6 days of culture; b: the average value of the wells with control culture medium added after 6 days of culture. The data were fitted using GraphPad Prism and the IC of the drug was calculated. 50 (Table 4).

[0309] The test showed that hRS7-MB3 showed a strong cytotoxic effect (IC 50 <1 nM), which was stronger than the control molecule hTINA1-DXd. In the human pharyngeal carcinoma squamous cell line FaDu, hRS7-MB3 showed comparable levels of inhibition to hTINA1-DXd, effectively inhibiting tumor cell growth (Figure 2A). Testing in the human pancreatic cancer cell line BxPC-3 revealed that hRS7-MB3 exhibited superior tumor suppression to the control molecule hTINA1-DXd (Figure 3A).

[0310] Compared to another positive control ADC molecule, Trodelvy, hRS7-MB3 demonstrated comparable efficacy against FaDu and BxPC-3 (Table 4), demonstrating potent tumor cell cytotoxicity (Figures 2B and 3B). However, the overall average DAR value for the control molecule, Trodelvy, was approximately 7.6, roughly double that of the present invention's hRS7-MB3. Therefore, it can be inferred that the present invention's ADC possesses stronger cytotoxicity.

[0311] 2. Bystander Effect

[0312] An important challenge in the treatment of solid tumors with antibody-drug conjugates (ADCs) is the heterogeneous expression of target antigens in tumor tissues or metastases, that is, cells with high, low, or no expression of the target antigen may coexist, thus affecting the efficacy of ADCs. The "bystander killing effect" has become a possible solution to this problem. ADCs can target cells with high TROP2 expression, kill target cells, and release loaded drugs. Some loaded drugs have good hydrophobic activity, and the released loaded drugs can pass through the cell membranes of cells surrounding the target cells, thereby achieving the effect of further killing tumor cells.

[0313] To verify the bystander effect of ADC targeting TROP2, two cell lines were used in this study, namely Colo-205 (Nanjing Kebai Biotechnology Co., Ltd.) and BxPC3 (ATCC). The culture medium used for both cells was RPMI 1640 (22400-071, Gibco) + 10% FBS (SH30406.05, HYCLONE). Colo-205 cells with low TROP2 expression were cultured with CellTrace TM Violet (C3455, Thermo) reagent was used for labeling at 37°C for 20 minutes, after which normal culture medium was added to terminate the reaction and washed twice with culture medium. Prepared BxPC3 and Colo-205 cells were added to a 24-well plate (NEST) at a ratio of 3:1, and ADC targeting TROP2 at a final concentration of 10 nM or an equal volume of culture medium was added to the corresponding wells, where IgG-MB3 was used as a negative control. The prepared samples were cultured in a 37°C incubator for 5 days. After the culture was completed, the cells were collected and stained with LIVE / DEAD TM The cells were stained with ELISA (Invitrogen, L34975) and analyzed by flow cytometry.

[0314] Testing revealed that Colo-205 cells, previously insensitive to the TROP2-targeting ADC, became responsive to the drug under a BxPC3-mediated "bystander effect." Compared to the control hTINA1-DXd, hRS7-MB3 produced a stronger bystander effect (Figure 4), further demonstrating that the toxin payload of the TROP2-targeting ADC of this application possesses stronger hydrophobic properties, enabling it to more effectively penetrate cell membranes and kill adjacent cells.

[0315] Effect Example 2: In Vivo Efficacy Verification of Anti-TROP2 Antibody-Drug Conjugate

[0316] 1. Mice: Immunodeficient mice CB17-SCID (Vitamin B) aged 6-8 weeks were placed in an SPF environment for 3-5 days before the experiment to allow them to better adapt to the current living environment.

[0317] In all experiments, the long and short diameters of the tumors were measured twice a week using a vernier caliper, and the tumor volume (mm) was calculated from the measured data. 3 The calculation formula is as follows: Tumor volume (mm 3 )=0.5*long diameter(mm)*short diameter(mm)*short diameter(mm)

[0318] 2. Resuspend human gastric cancer cell line NCI-N87 (ATCC) cells in 1x PBS (Gibco, 10010049) at an appropriate concentration and culture at 2x10 6 The tumor cells were implanted subcutaneously on the right side of the mouse abdomen. 3 The mice were grouped so that the average initial dosing volume of each group was basically the same. After grouping, different doses of antibody-drug conjugates hRs7-DXd, hRS7-MB3, hTINA1-DXd, or control conjugates IgG-MB3, IgG-DXd and IgG control were administered by intraperitoneal injection. After administration, it can be seen that compared with the control antibody-drug conjugates (IgG-MB3, IgG-DXd), the two groups of low-dose (1 mg / kg) TROP2-targeted antibody-drug conjugates hRS7-DXd, hTINA1-DXd and hRS7-MB3 all exerted a certain degree of tumor inhibitory effect (Figure 5A), and their tumor inhibitory effects were similar. In the high-dose group, hRS7-DXd and hTINA1-DXd showed comparable tumor inhibitory effects, indicating that the two TROP2 antibodies have equivalent endocytic activity. Compared with hRS7-DXd, hRS7-MB3 has a stronger tumor inhibitory effect. Finally, compared with Daiichi Sankyo's TROP2-ADC (hTINA1-DXd), hRS7-MB3 at the same dose (3 mg / kg) exhibited a stronger and more significant tumor inhibitory effect ( Figure 5B ), suggesting that hRS7-MB3 may have best-in-class activity.

[0319] 3. HCC827 cells, a non-small cell lung cancer cell line purchased from ATCC, were inoculated subcutaneously into mice (2x10 6 / mouse), and wait until the tumor volume grows to 120-150 mm 3 The mice were grouped and dosed. Different doses of antibody-drug conjugates and control drugs were injected into the mice by intraperitoneal administration. The entire experiment was administered only once, and the growth status of the mice was observed and the changes in the tumor volume of the mice were measured. After administration, it was found that the high-dose group (5 mg / kg) antibody-drug conjugates showed a strong and equivalent tumor inhibitory effect (Figure 6). In the low-dose group (2 mg / kg), both groups of ADCs targeting TROP2 exerted tumor proliferation inhibitory effects, but compared with hTINA1-DXd (DS-1062), hRS7-MB3 showed a more significant tumor inhibitory effect (Figure 6).

[0320] 4. The lung squamous cell carcinoma cell line LK-2 (CBP60105) purchased from Nanjing Kebai Biotechnology was cultured at 3x10 6The tumor cells were implanted subcutaneously on the right side of the mouse abdomen. 3 The mice were grouped so that the average initial dosing volume for each group was essentially the same. The entire experiment was administered only once, with 10 mg / kg injected into the mice via intraperitoneal administration. IgG-MB3 or IgG-DXd served as negative controls. Observations revealed that in tumor models with relatively low TROP2 expression, the two ADCs targeting TROP2 were also able to delay tumor growth (Figure 7). Compared to hTINA1-DXd (DS-1062), hRS7-MB3 showed a better tumor inhibitory effect.

[0321] 5. Pancreatic cancer cell line BxPC3 cells purchased from ATCC were cultured at 3x10 6 The tumor cells were implanted subcutaneously on the right side of the abdomen of mice. On day 8, the tumor volume grew to 150-180 mm. 3 The mice were grouped to ensure that the average initial dosing volume of each group was basically the same. The grouped mice were treated with 3 mg / kg and 10 mg / kg doses by intraperitoneal injection, and only one dose was given throughout the experiment. The experimental group was given ADCs targeting TROP2 (hTINA1-DXd and hRS7-MB3), and the control group was treated with the same amount of IgG-MB3 or IgG-DXd. As can be seen from the results (Figure 8), at a low dose (3 mg / kg), hRS7-MB3 and hTINA1-DXd (DS-1062) showed equivalent tumor inhibitory effects. In the high-dose group (10 mg / kg), hRS7-MB3 showed a more significant tumor inhibitory effect than hTINA1-DXd. On day 38, the tumor volume of the hRS7-MB3 group was significantly lower than that of the hTINA1-DXd group (P=0.008). Judging from the overall tumor growth curve, 3 mg / kg of hRS7-MB3 and 10 mg / kg of hTINA1-DXd showed the same tumor inhibitory effect. From this, we can infer that hRS7-MB3 may have better performance than hTINA1-DXd in clinical practice.

[0322] 6. The lung squamous cell carcinoma cell line EBC-1 (JCRB0820) cells purchased from JCRB cell bank were cultured at 1x10 6 / Mouse inoculation amount Tumor cells were implanted subcutaneously on the right side of the mouse abdomen. When the tumor volume grew to 150-180mm 3Mice were grouped to ensure that the average initial dose volume for each group was roughly the same. The grouped mice were then administered a single dose of 1 mg / kg via intraperitoneal injection. As shown in Figure 9, hRS7-MB3 exhibited a very strong tumor-suppressing effect at this low dose.

[0323] Trodelvy is an approved TROP2-ADC molecule. Preclinical and clinical data show that Trodelvy's linker-payload has poor stability. Trodelvy is poorly stable in mice, with a half-life of 11 hours (Bioconjugate Chem. 2015, 26, 919-931), resulting in a higher dosing frequency (once a week) required in the clinic to maintain drug concentrations in the blood. Therefore, we infer that in an in vivo mouse tumor model, a single dose of hRS7-MB3 has a better tumor inhibitory effect than a single dose of Trodelvy.

[0324] Effect Example 3: ADC in vivo stability test in mice

[0325] 1. Balb / c mice (Weitonglihua) were injected with the antibody-drug conjugate via the tail vein at a dose of 10 mg / kg. Blood was collected at 0.083 h, 0.5 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, 336 h, and 504 h after administration (Figure 10A). After standing at room temperature, the serum was centrifuged to test the drug stability in mice.

[0326] 2. Antibody Detection: Coat the plate with TROP2 protein (ACRO, cat: TR2-H5223, lot: 2573-2316F1-1B9) and incubate overnight at 4°C (Figure 10A). Remove the coating solution and add 300 μL of wash buffer (0.05% Tween 20) to each well, washing three times. Add 200 μL of blocking buffer (5% skim milk powder) to each well and block at room temperature for 2 hours. Discard the blocking buffer and add 300 μL of wash buffer to each well, washing three times. Add the diluted standards, quality control samples, and the mouse serum samples to be tested to the plate and incubate at room temperature for 2 hours. Discard the sample solution and add 300 μL of wash buffer to each well, washing three times. Dilute anti-hFc-HRP (BETHYL, cat: A80-104P, lot: 97) 20,000-fold and add 100 μL to each well, incubating at room temperature for 1 hour. Pour off the liquid and add 300 μL of wash buffer to each well. Repeat the wash six times. Add 100 μL of TMB substrate (Solarbio, cat: PR1200, lot: 20230418) to each well and develop color at room temperature in the dark for 5–10 minutes. Add 50 μL of ELISA stop buffer (Beijing Solaibao C1058 20190620) to each well and shake at medium speed for 10 seconds. Within 30 minutes, read the OD at 450 nm / 620 nm in a microplate reader (Thermo Multiskan FC AS-A1-008, USA).

[0327] 3. Antibody-Drug Conjugate ADC Detection: Coat the plate with the anti-payload antibody 18E7C9 (Innovent, lot: 20220424) and incubate overnight at 4°C (Figure 10A). Discard the coating protein solution, add 300 μL of wash solution (0.05% Tween 20) to each well, and wash three times. Add 200 μL of blocking solution (5% skim milk powder) to each well and block at room temperature for 2 hours. Discard the blocking solution, add 300 μL of wash solution to each well, and wash three times. Add the diluted standards, quality control samples, and the mouse serum sample to be tested to the plate and incubate at room temperature for 2 hours. Discard the sample solution, add 300 μL of wash solution to each well, and wash three times. Add the biotinylated TROP2 protein (ACRO, cat: TR2-H82E5, lot: BV3055-91PF1-ZC) to the ELISA plate and incubate at room temperature for 2 hours. Discard the liquid and add 300 μL of wash buffer to each well, repeating the wash three times. Dilute SA-HRP (Biolegend, cat: 405210, lot: B339457) 20,000-fold and add 100 μL to each well. Incubate at room temperature for 1 hour. Discard the liquid and add 300 μL of wash buffer to each well, repeating the wash six times. Add 100 μL of TMB substrate (Solarbio, cat: PR1200, lot: 20230418) to each well and develop color at room temperature in the dark for 5-10 minutes. Add 50 μL of ELISA stop buffer (Beijing Solaibao C1058 20190620) to each well, shake at medium speed for 10 seconds, and read the OD 450 nm / 620 nm value in a microplate reader (Thermo Multiskan FC AS-A1-008, USA) within 30 minutes.

[0328] After testing, it was found that the half-life (T-half) of hRS7-MB3 was 197.74h, which is 154.59h compared to the half-life of the control ADC molecule hTINA1-DXd (Table 5). hRS7-MB3 has better stability in mice as a whole. The overall exposure (AUC) of hRS7-MB3 in mice is also higher than that of hTINA1-DXd (Table 5, Figure 10B). From the perspective of the clearance rate (Cl_obs) of the overall ADC molecule, hRS7-MB3 is 0.59, which is much lower than the 0.83 of the control molecule hTINA1-DXd (Table 5). Compared with the half-life of the antibody, the linker-load in hRS7-MB3 also has good stability (Figure 10B). The approved positive control ADC molecule, Trodelvy, has a half-life of 11 hours in mice (at 10 mg / kg) (Bioconjugate Chem. 2015, 26, 919-931), significantly lower than that of hRS7-MB3. In summary, hRS7-MB3 exhibits superior stability in mice compared to the two positive control ADC molecules.

[0329] Table 5: Stability parameters of ADC in mice

[0330] Effect Example 4: Safety Evaluation of Antibody-Drug Conjugates

[0331] In order to evaluate the safety of antibody-drug conjugates, we selected crab-eating macaques, a species that cross-reacts with antibodies, for experiments in GLP toxicology. Specifically, the antibody-drug conjugate hRS7-MB3 was divided into three dosing groups: 20 mg / kg, 50 mg / kg, and 100 mg / kg. Each group consisted of 10 monkeys (5 males and 5 females), and the drug was administered once every three weeks for a total of 3 times. After the end of the dosing, the observation was continued for 1-3 weeks. As a result, the highest non-severe toxic dose (HNSTD) of the antibody-drug conjugate hRS7-MB3 was 100 mg / kg.

[0332] In contrast, the positive control ADC molecule DS-1062 (Daiichi Sankyo) has a HNSTD of 30 mg / kg (WO2020240467A1) at the same dosing frequency (once every three weeks, a total of 3 times), which is much lower than hRS7-MB3. In addition, another positive control ADC molecule Trodelvy disclosed in the document (EMEA / H / C / 005182 / 0000) HNSTD (Day 1 and Day 8 / Every 21 days, a total of 4 cycles) is 50 mg / kg. In summary, it can be seen that hRS7-MB3 has better tolerability in monkeys and may have a higher tolerated dose clinically. Combined with the in vivo and in vitro efficacy experimental results mentioned above, we can infer that hRS7-MB3 has a wider therapeutic window in the clinic.

Claims

1. An antibody-drug conjugate represented by the following formula (I), its stereoisomer or pharmaceutically acceptable salt or solvate, in, D is shown in the following formula (II): Among them, R 1 is selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C2-C6 haloalkenyl and C2-C6 haloalkynyl; R 2 Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 6 and-SR 6 ; R 3 Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 6 ; or R 2 and R 3 Together they form -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2; R 6 is selected from H or C1-C4 alkyl; and L1 is -L 1b -L 1a -, where L 1b Connect with Q', L 1a Connect with D, Where L 1a is not present or -(C1-C 10 Alkylene)-; L 1b Does not exist, *-(C1-C 10 Alkylene)-C(O)N(R 5 )-or*-(C1-C 10 Alkylene)-N(R 5 )C(O)-; wherein * indicates that the terminus is covalently linked to Q'; and R 5 is H or C1-C6 alkyl, Q' is -O- or -S-; E is -CH2-NH-M, wherein M is a peptide residue comprising 2 to 10 amino acids; wherein the peptide residue is optionally substituted by one or more groups independently selected from C 1-6 An alkyl group and a polyol group; and wherein the N-terminus of M is covalently attached to Z'; Z' is -C(=O)-L2-Y'-, wherein Z' is covalently linked to E via the -C(=O) moiety shown and to A via the Y' moiety; L2 is selected from -(CH2) m -(O-CH2CH2) m1 -(CH2) m2 -、-(CH2) m -(O-CH2CH2) m1 -NHC(O)-(CH2) m -、 -(CH2) m -(O-CH2CH2) m1 -C(O)NH-(CH2) m -, wherein m1 and m2 are independently selected from integers of 0-20; m is selected from integers of 1-10, wherein the left end of these groups is connected to -C(=O)-, and the right end is connected to Y'; Y' is composed of The groups formed; A represents an antibody or antibody fragment targeting TROP2, comprising a heavy chain and a light chain, wherein the heavy chain comprises a CDR1 comprising or consisting of a sequence shown in SEQ ID NO: 1, a CDR2 comprising or consisting of a sequence shown in SEQ ID NO: 2, and a CDR3 comprising or consisting of a sequence shown in SEQ ID NO: 3, The light chain comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6; and p is the average value of the drug to antibody ratio and is a value between 1-15, for example, a value between 3-5, 3.5-4.

5.

2. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the antibody or antibody fragment targeting TROP2 comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has at least 80% sequence identity, preferably at least 90% sequence identity, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with the amino acid sequence SEQ ID NO:7, and the light chain variable domain has at least 80% sequence identity, preferably at least 90% sequence identity, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity with the amino acid sequence SEQ ID NO:

8.

3. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein the antibody or antibody fragment targeting TROP2 comprises a heavy chain variable domain and a light chain variable domain, wherein the light chain variable domain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:8; and the heavy chain variable domain comprises, or consists of, the amino acid sequence shown in SEQ ID NO:

7.

4. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof of any one of claims 1 to 3, wherein the antibody or antibody fragment targeting TROP2 comprises a heavy chain and a light chain, wherein the light chain of the antibody targeting TROP2 or its fragment has at least 80% sequence identity with the amino acid sequence SEQ ID NO: 10, preferably at least 90% sequence identity, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity; and the heavy chain of the antibody targeting TROP2 or its fragment has at least 80% sequence identity with the amino acid sequence SEQ ID NO: 9, preferably at least 90% sequence identity, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% sequence identity.

5. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, wherein the antibody or antibody fragment targeting TROP2 comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:9, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

10.

6. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the antibody fragment is selected from Fab, Fab', F(ab')2, Fab'-SH, scFv or scFv-Fc. 7 . The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6 , wherein the antibody targeting TROP2 is a monoclonal antibody.

8. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the antibody targeting TROP2 is a chimeric antibody or a humanized antibody.

9. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 8, in, R 1 Selected from -H or C1-C4 alkyl; R 2 is selected from -H, -F, C1-C3 alkyl or C1-C3 haloalkyl; R 3 Selected from -H, -F, -CN, -OCH3, -CH3 or -CF3; L1 is independently unsubstituted or halogenated -(C1-C 10 Alkylene)-; Q' is -O- or -S-; E is -CH2-NH-M, wherein M is a peptide comprising 2 to 10 amino acids; wherein optionally the amino acids are substituted with one or more polyols; and wherein the N-terminus of M is covalently attached to Z'; Z' is -C(=O)-L2-Y'; L2 is -(C1-C 10 Alkylene)-.

10. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 9, wherein: Y' is Wherein * indicates the site of covalent bonding to the A.

11. The antibody-drug conjugate according to any one of claims 1 to 10, its stereoisomer or pharmaceutically acceptable salt or solvate, Where L2 is -(CH2) m -, m is an integer selected from 1-10.

12. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 11, wherein Z' is formed by:

13. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 12, wherein Z' is: Where * indicates the site of covalent attachment to A.

14. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 13, wherein: The M is a peptide comprising 2, 3 or 4 amino acids; Each amino acid of M is an L amino acid; Or at least one amino acid of M is a D amino acid.

15. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 13, wherein the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, tryptophan, aspartic acid, asparagine, isoleucine, arginine and proline, and wherein the glutamine or glutamic acid is optionally substituted with 1 polyol group and optionally substituted with 1 C 1-6 Alkyl substitution.

16. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 15, wherein: The M comprises one or more amino acids selected from the group consisting of glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, and wherein the glutamine or glutamic acid is optionally substituted with a polyol.

17. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 15, The substituted glutamine or glutamic acid has the structure shown below: Where R 4 is H or C1-C6 alkyl; Preferably Where R 4 It is H or C1-C6 alkyl.

18. The antibody-drug conjugate according to claim 17, its stereoisomer or pharmaceutically acceptable salt or solvate, The substituted glutamine or glutamic acid has the structure shown below: Where R 4 is H or C1-C6 alkyl; Preferably Where R 4 It is H or C1-C6 alkyl.

19. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 17, wherein: M comprises amino acids having the following structure, Where R 4 It is -H or C1-C6 alkyl.

20. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 19, wherein: M comprises amino acids having the structure 21. The antibody-drug conjugate, stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 20, wherein M is selected from -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Leu-Ala-*, -Ala-Leu-*, -Ala-Ala-*, -Phe -Gln-*, -Gln-Phe-*, -Leu-Gln-*, -Gln-Leu-*, -Val-Gln-*, -Phe-Ala-*, -Ala-Phe-*, -Gln-Val-*, -Ala-Ala-Ala-*, -Gly-Gly-Gly-*, -Ala-Val-Ala-*, -Gly-Val-Gly-*, -Ala-Val-Gly-*, -Gly-Phe-Gly-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Gln-*, -Ala-Val-Ala-Gly-*, -Gly-Phe-Gly-Gly-*, -Gly-Gly-Phe-Gly-*, -Gly-Phe-Gly-Gln-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala-*, -Ala-Val-Ala-Ala-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, -Gly-Phe-Gly-Gln-*, and -Gly-Leu-Phe-Gly-*, where * indicates the N-terminus of the peptide to which Z' is covalently attached.

22. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 20, wherein The M is selected from -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-*, -L-Ala-D-Val-L-Ala-*, -L-Ala-D-Ala-Gly-*, -L-Ala-D-Val-Gly-*, -L-Ala-L-Val-Gly-*, -L-Ala-L-Val-L-Gln-*, or -Gly-L-Phe-Gly-L-Gln-*, wherein * indicates the N-terminus of the peptide to which Z' is covalently attached.

23. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 22, wherein: -E- is selected from the following structures, wherein * represents the N-terminus of the peptide to which Z' is covalently attached:

24. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein: Z'-E is formed by one of the following structures: Alternatively, Z'-E is selected from the following structures, where * indicates the point of attachment to A:

25. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 24, wherein: D is shown in the following structure:

26. The antibody-drug conjugate, stereoisomer, or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 25, wherein: D-L1-Q'- has one of the following structures:

27. The antibody-drug conjugate, stereoisomer, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 26, wherein the antibody-drug conjugate has the following structure: wherein A and p are as defined in any one of claims 1-26.

28. An antibody-drug conjugate represented by formula (I'), a stereoisomer or a pharmaceutically acceptable salt or solvate thereof, wherein q is DAR, and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and A, Z', E, Q', L1 and D as defined in any one of claims 1-27.

29. A pharmaceutical composition comprising the stereoisomer or pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 28, and optionally a pharmaceutically acceptable carrier.

30. A pharmaceutical combination comprising a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 28, and one or more other therapeutic agents, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

31. Use of the antibody-drug conjugate according to any one of claims 1 to 28, its stereoisomer or pharmaceutically acceptable salt or solvate, the pharmaceutical composition according to claim 29 or the pharmaceutical combination according to claim 30 in the preparation of a medicament for treating or preventing a cell proliferative disease or disorder or inhibiting abnormal cell growth, preferably the cell proliferative disease or disorder is cancer.

32. The use according to claim 31, wherein The cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colon or colorectal cancer, diffuse intrinsic pontine glioma, endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, nasopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer or Wilms' tumor, preferably, the cancer is a TROP2-positive cancer.

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