Anti-TROP2 Antibodies and Their Conjugates

Abstract

The present disclosure relates to antibodies, payloads, and linker molecules for targeting molecular-drug conjugates, as well as corresponding conjugates, their preparation, and use.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceuticals, and in particular, to antibodies, payloads and linkers for preparing targeted molecule-drug conjugates, as well as corresponding conjugates, their preparation processes, and their uses.

Background Art

[0002] TROP2 is a transmembrane protein and has been found to be overexpressed in several cancer types, including endometrial cancer, prostate cancer, pancreatic cancer, colon cancer, gastric cancer, oral cancer, and glioma, making it a natural candidate for the development of targeted therapies. TROP2 acts as a regulator of cell self-renewal, proliferation, and transformation. Experiments have shown that TROP2 can promote tumor growth and that tumor cell proliferation is inhibited when the TROP2 gene is knocked out. Since the tissue expression of TROP2 is limited, the toxicity of treatment is reduced, which is also an advantage of therapies targeting TROP2. Multiple ADCs targeting TROP2 have been proposed (for example, DS-1062, TRODELVY, BAT8003). These therapeutic agents have significantly improved the survival rate of patients with TROP2-positive cancers.

[0003] DS-1062 is an antibody-conjugate drug developed by Daiichi Sankyo using its own DXd ADC technology. It consists of a monoclonal antibody that targets the Trop2 protein conjugated to DXd. Data including more NSCLC patients have shown that DS-1062 exhibits good dose-dependent anti-cancer activity. As the dose increased, tumors in more NSCLC patients shrank. TRODELVY is the first FDA-approved ADC specific for recurrent or refractory metastatic TNBC and the first anti-TROP2 ADC approved by the FDA. It consists of an antibody that targets TROP2 conjugated to SN-38, an active metabolite of the chemotherapeutic drug irinotecan.

[0004] However, there is still a high demand for new ADC drugs that target TROP2, and safe ADCs are also one direction for the development of new drugs. Summary of the Invention

[0005] In a first aspect, the compound of formula (I): The compound TIFF2025524725000001.tif33165 is provided. During the ceremony, W is hydrogen, LKb, or -C2H4-(PEG) t -(CO)NH2, Y is hydrogen or LKa-LKb; However, W and Y cannot be hydrogen at the same time, Each LKa independently: Selected from TIFF2025524725000002.tif25165, opSu TIFF2025524725000003.tif30165 or a mixture thereof, Each LKb is independently 2 -L 1 -B, Each B independently represents a terminal group R 10 Or (1), (2), and (3) below: (1) self-immolative spacer Sp1, (2) bond, or -CR 1 R 2 -, C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 (3) a terminal group R 10 It is a combination of R 10 is hydrogen or a group that can be eliminated when reacting with a group of the payload, L 1 is cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and cleavable sequence 1 contains 1 to 10 amino acids; L 2is a bond, or C 2-20 is alkylene, and one or more -CH 2- structures in the alkylene are -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, TIFF2025524725000004.tif12165C 4-10 cycloalkylene, C 4-10 heterocyclylene, or phenylene, which may be replaced, where cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or substituted with at least one substituent selected from halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R 9 and are substituted with at least one substituent selected from the group consisting of: Ld2 and each Ld1 are independently a bond or selected from -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-, or are independently an unsubstituted natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10 and a side chain substituted with -(PEG) j -R 11 ; -(PEG) t -, -(PEG) i -, and -(PEG) j - are each a PEG fragment containing the indicated number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, and having an optional additional C 1-10 alkylene at one end; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently hydrogen, halogen, -C 1-10Alkyl, -C 1-10 Haloalkyl, C 4-10 Selected from cycloalkylene, or R 1 And R 2 Together with the carbon atom to which they are attached, form a 3- to 6-membered cycloalkyl group, or R 3 And R 4 Together with the carbon atom to which they are attached, form a 3- to 6-membered cycloalkyl group, and R 11 Is C 1-10 Alkyl, and m is any integer from 1 to 3, n is any integer from 2 to 20, d is 0 or any integer from 1 to 6, each i is independently an integer from 0 to 100, preferably 0 to 20, preferably each i is independently an integer from 0 to 12, more preferably 0 to 8, particularly 4, Each j is independently an integer from 1 to 100, preferably 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably 8 to 12, particularly 8 or 12, Each t is independently an integer from 1 to 100, preferably 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably 8 to 12, particularly 8 or 12.

[0006] In a second aspect, a compound having the structure of formula (II): TIFF2025524725000005.tif32165 is provided, Wherein, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P, M is hydrogen or LKa-LKb-P, Provided that Q and M are not simultaneously hydrogen, P is the payload attached to the B moiety or L 1 Moiety of the compound of formula (I), n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I). Preferably, M is hydrogen or LKa-L 2 -L 1 -B-P, where each B is independently absent or one or more combinations of the following divalent groups (1) and (2): (1) a self-destructive spacer Sp1, and (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and -(CO)-, one or a combination of two or more of the selected divalent groups is a combination of Preferably, Sp1 is selected from PABC, acetal, heteroacetal, and combinations thereof, more preferably, Sp1 is acetal, heteroacetal, or PABC, still more preferably, the heteroacetal is selected from N,O-heteroacetal, and more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, where -O- or -NH- is bonded to the cleavable sequence 1, and U is absent or O, S, or NH, preferably O or S.

[0007] In a third aspect, an anti-TROP2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (V H ) and a light chain variable region (V L ) is provided, V H is (i) HCDR1 comprising the amino acid sequence of SX1AGMN (where X1 is N or A), (ii) HCDR2 comprising the amino acid sequence of WINTDSGEPTYTDDFKG (SEQ ID NO: 10) or WINTYTGEPTYTDDFKG (SEQ ID NO: 8), and (iii) HCDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 11) is included, and / or V L is (i) LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14), (ii) LCDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 15), and (iii) LCDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) is included.

[0008] In a fourth aspect, formula (i): A cytotoxin having the structure of TIFF2025524725000006.tif68165 is provided, wherein, a * is 0 or 1, p1 * and p2 * The carbon atoms marked with are each an asymmetric center, and the asymmetric center is in the S configuration, R configuration, or racemic form, L 1* is unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH2, C 1-6 selected from alkylene, M * is -CH2-, -NH-, or -O-, L 2* is C 1-3 alkylene, R 1* and R 2* are each independently selected from hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0009] In a fifth aspect, formula (III): A conjugate having the structure of TIFF2025524725000007.tif37165 is provided, wherein, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb―P, M is hydrogen or LKa-LKb―P, Each LKa is independently, Selected from TIFF2025524725000008.tif27165, opSu is TIFF2025524725000009.tif32165 or a mixture thereof, each LKb is independently L 2 -L 1 -B, m, n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I), each B is independently absent or one or more combinations of the following (1) and (2): (1) a self-destructive spacer Sp1, and (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-, preferably, B is -NH-CH2-U, absent, -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, or NH-CH2-U-(CH2) g -(CO)-, provided that Q and M are not simultaneously hydrogen, P is a payload that binds to the B moiety or the L 1 moiety of the compound of formula (I), each L 1 is independently a cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1 to 10 amino acids, each L 2 is independently a bond or C 2-20 alkylene, and one or more -CH2- structures in the alkylene are -CR 3 R 4 -, -O-, -(CO)-, -S(=O) 2- -, -NR 5 -, TIFF2025524725000010.tif17165C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, or phenylene, each of which may be independently unsubstituted or substituted with at least one substituent selected from halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R 9 and is substituted with at least one substituent selected from the group consisting of: -(PEG) t -, -(PEG) i - and -(PEG) j - each represents a PEG fragment containing the indicated number of consecutive -(O-C2H4)- or -(C2H4-O)- structural units, with an optional additional C 1-10 alkylene at one end; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, C 4-10 cycloalkylene, or R 1 and R 2 together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl group, or R 3 and R 4 together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl group; each i is independently an integer from 0 to 100, preferably from 0 to 20, more preferably each i is independently an integer from 0 to 12, even more preferably from 0 to 8, and particularly 4; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20, and preferably each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, particularly 8 or 12, Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20, and preferably each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, particularly 8 or 12, Each g is independently an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, A is an anti-TROP2 antibody or an antigen-binding fragment thereof that binds to the G n moiety of the compound of formula (I), and G is glycine, z is an integer from 1 to 20.

[0010] In a sixth aspect, there is provided the use of an anti-TROP2 antibody or an antigen-binding fragment thereof or a pharmaceutical composition in the manufacture of a medicament for preventing or treating a disease, wherein the disease is a tumor.

[0011] In a seventh aspect, there is provided a polynucleotide encoding an antibody or an antigen-binding fragment thereof.

[0012] In an eighth aspect, there is provided a vector comprising the polynucleotide.

[0013] In a ninth aspect, there is provided a host cell comprising the polynucleotide or the vector.

[0014] In a tenth aspect, there is provided a method for producing the antibody or an antigen-binding fragment thereof, comprising: (i) culturing a host cell under conditions suitable for the expression of the antibody or an antigen-binding fragment thereof; and (ii) recovering the antibody or an antigen-binding fragment thereof from the host cell or its cell culture. The method is provided.

[0015] In another aspect, there is provided a pharmaceutical composition comprising a prophylactically effective amount or a therapeutically effective amount of a conjugate and at least one pharmaceutically acceptable carrier.

[0016] In another aspect, there is provided a pharmaceutical composition comprising a prophylactically effective amount or a therapeutically effective amount of an antibody or antigen-binding fragment and at least one pharmaceutically acceptable carrier.

[0017] In another aspect, there is provided the use of the conjugate, the antibody, or the pharmaceutical composition in the manufacture of a medicament for preventing or treating a disease, wherein the disease is a tumor.

[0018] In another aspect, there is provided a method of treating a subject suffering from a disease or preventing disease progression, the method comprising administering the conjugate, the antibody, or the pharmaceutical composition, wherein the disease is a tumor.

[0019] In one embodiment, the tumor is a TROP2-related tumor.

[0020] In some embodiments, the disease is a tumor. In some embodiments, the disease comprises a TROP2-positive tumor. In some embodiments, the disease includes tumors that overexpress TROP2 or tumors having a TROP2 gene mutation. In some embodiments, the disease is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma. In one embodiment, the TROP2-related tumor is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial cancer.

[0021] In another aspect, there is provided an antibody-drug conjugate comprising the antibody.

Brief Description of the Drawings

[0022]

Figure 1.1

Figure 1.2

Figure 1.3

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13.1

Figure 13.2

Figure 14

Figure 15

Figure 16

Figure 17

Mode for Carrying Out the Invention

[0023] Detailed Description Specific embodiments are provided below to explain the technical content of the present disclosure. Those skilled in the art can easily understand other advantages and effects of the present disclosure through the content disclosed in this specification. The present disclosure can also be implemented or applied through other different specific embodiments. Without departing from the spirit of the present disclosure, those skilled in the art can make various modifications and changes.

[0024] Definitions Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The techniques used herein refer to techniques commonly understood in the relevant art, including obvious variations and equivalent substitutions for one of ordinary skill in the art. The following terms are considered to be readily understandable by one of ordinary skill in the art, but the following definitions are provided to more fully explain the present disclosure. When a trade name exists in this specification, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0025] It should be understood that when a particular quantity, concentration, or other value or parameter is presented in the form of a range, preferred range, or preferred upper or lower limit, this is equivalent to specifically disclosing any range formed by combining any upper limit value or preferred value with any lower limit value or preferred value (regardless of whether the range is explicitly recited). Unless otherwise specified, numerical ranges recited herein are intended to include the endpoints of the range, as well as all integers and fractions (decimals) within the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The same understanding applies to other similar expressions, such as for j, k, and g.

[0026] Unless the context clearly indicates otherwise, the singular forms "a" and "the" include the plural. The expressions "one or more" or "at least one" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0027] The terms "about" and "approximately" when used in connection with a numerical variable generally mean that the value of that variable and all values of that variable are within experimental error (e.g., within a 95% confidence interval of the mean value), or within ±10% of the specified value, or within a wider range.

[0028] The term "stoichiometric ratio" means adapting various substances according to the amounts per specific weight. For example, in the present disclosure, the active ingredient is mixed with an injection agent, a binder, and a lubricant in a specified weight ratio.

[0029] The term "optional" or "optionally" means that the event described following that term may not necessarily occur, and the description includes cases where the event or situation occurs or does not occur.

[0030] The expressions "comprising", "including", "containing", and "having" are open-ended and do not exclude additional unenumerated elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of" is limited to optional elements, steps, or components that, in addition to the specified elements, steps, or components, do not substantially affect the essential and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0031] As used herein, the term "antibody" is used in a broad sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments only if they have the desired biological activity. Antibodies can be of any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and can be derived from any suitable species. In some embodiments, the antibodies are of human or murine origin. Antibodies can also be fully human antibodies, humanized antibodies, or chimeric antibodies prepared by recombinant methods.

[0032] As used herein, monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for a few possible natural variations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" indicates that the antibody characteristics are obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular antibody production method.

[0033] An intact antibody or full-length antibody essentially comprises antigen-binding variable region(s), as well as light chain constant region(s) (CL) and heavy chain constant region(s) (CH) (CH1, CH2, CH3, and CH4 may be included depending on the antibody subtype). The antigen-binding variable region (also known as the fragment variable region, Fv fragment) usually includes a light chain variable region (VL) and a heavy chain variable region (VH). The constant region can be a constant region having a native sequence (e.g., a constant region having a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0034] An antibody fragment can comprise a portion of an intact antibody, preferably its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs). A Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin or a fragment having the same structure produced, for example, by recombinant expression. A Fab fragment contains a light chain (including VL and CL) and another chain, which contains the variable domain of the heavy chain (VH) and the constant region domain of the heavy chain (CH1). An F(ab’)2 fragment is an antibody fragment obtained by pepsin digestion of an immunoglobulin (pH 4.0 - 4.5) or a fragment having the same structure produced, for example, by recombinant expression. An F(ab’)2 fragment essentially contains two Fab fragments, and each heavy chain portion contains several additional amino acids including cysteine that forms a disulfide bond binding the two fragments. A Fab’ fragment is a fragment containing half of an F(ab’)2 fragment (one heavy chain and one light chain). An antibody fragment can contain multiple chains joined together, for example, via disulfide bonds and / or via a peptide linker. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd’ fragments, and other fragments including modified fragments. An antibody fragment usually contains at least or about 50 amino acids, usually at least or about 200 amino acids. An antigen-binding fragment can include any antibody fragment that can result in an antibody that binds immunospecifically to an antigen when inserted into an antibody framework (e.g., by substitution of the corresponding region).

[0035] Antibodies according to the present disclosure can be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimetics) can be expressed by appropriate culture systems (e.g., E. coli or mammalian cells). This manipulation can refer to, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0036] Cytotoxins refer to substances that inhibit or prevent the expression activity and cell functions of cells and / or cause cell destruction. The cytotoxins currently used in ADCs are more toxic than chemotherapeutic drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, regulation of apoptosis. Drugs targeting the microtubule cytoskeleton can be, for example, microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, mitansine, auristatin, vinblastine, colchicine, and dolastatin. DNA-targeting drugs can be, for example, drugs that directly disrupt the DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt the DNA structure include, but are not limited to, DNA double-strand breakers, DNA alkylating agents, DNA intercalators. DNA double-strand breakers can be, for example, enediyne antibiotics such as dynemicin, esperamicin, neocarzinostatin, and uncialamycin, etc., but are not limited thereto. DNA alkylating agents can be, for example, DNA bis-alkylating agents (i.e., DNA cross-linkers) or DNA mono-alkylating agents. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, dihydroindole benzodiazepine (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2), the structures of which are shown below), camptothecin, and anthracyclines. RNA-targeting drugs can be, for example, drugs that inhibit splicing, and examples thereof include, but are not limited to, pladienolide.Drugs targeting kinesin-mediated protein transport can be mitotic kinesin inhibitors, including, but not limited to, for example, kinesin spindle protein (KSP) inhibitors.

[0037] A spacer is a structure located between different structural modules and can spatially separate the structural modules. The definition of a spacer is not limited by whether it has a specific function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, and the non-amino acid structures can be, but are not limited to, amino acid derivatives or analogs. A "spacer sequence" refers to an amino acid sequence that functions as a spacer, and examples thereof include a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids such as GA, or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc., but are not limited thereto. A self-destructive spacer is a covalent assembly adjusted to correlate the cleavage of two chemical bonds after activation of the protecting moiety in the precursor: upon stimulation, the protecting moiety (e.g., a cleavable sequence) is removed, triggering a series of degradation reactions and the sequential release of smaller molecules over time. Examples of self-destructive spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0038] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is bonded to the remainder of the molecule via a single bond. The alkyl group can contain 1 to 20 carbon atoms, C1-C 20An alkyl group, for example, a C1-C4 alkyl group, a C1-C3 alkyl group, a C1-C2 alkyl, a C3 alkyl, a C4 alkyl, a C3-C6 alkyl is meant. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof, but are not limited thereto. A divalent radical refers to a group obtained from the corresponding monovalent radical by removing one hydrogen atom from a carbon atom by a free valence electron(s). A divalent radical has two bonding sites bonded to the remainder of the molecule. For example, "alkylene" or "alkylidene" refers to a saturated divalent hydrocarbon group that is either straight-chain or branched. Examples of alkylene groups include methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, etc., but are not limited thereto.

[0039] As used herein, when a group is combined with another group, the bond of the group can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination can be bonded to other parts of the molecule via any suitable atom within the structure, preferably via a specified chemical bond. For example, -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10When two or more of the divalent groups selected from heterocyclylene and -(CO)- combine together to form a combination, two or more of the divalent groups are linearly bonded to each other, for example, -CR 1 R 2 -C 1-10 alkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-C 1-10 alkylene-(CO)-, -CR 1 R 2 -CR 1’ R 2’ -(CO)-, -CR 1 R 2 -CR 1’ R 2’ -CR 1’’ R 2’’ -(CO)- and the like can be formed. The resulting divalent structure can further be bonded to other parts of the molecule.

[0040] The expressions "antibody-conjugated drug" and "antibody-drug conjugate" have the same meaning when used herein.

[0041] Compound of formula (I) In one embodiment, formula (I): Compounds of TIFF2025524725000011.tif32165 are provided, wherein W is hydrogen, LKb, or -C2H4-(PEG) t -(CO)NH2, [[ID=XY]] Y is hydrogen or LKa-LKb, provided that W and Y are not hydrogen at the same time, each LKa is independently selected from TIFF2025524725000012.tif26165, opSu is TIFF2025524725000013.tif32165 or a mixture thereof, Each LKb is independently, L 2 -L 1 -B, and each B is independently a terminal group R 10 or one of the following (1), (2), and (3): (1) a self-destructive spacer Sp1, (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and one or a combination of two or more of the divalent groups selected from -(CO)-, and (3) a combination of a terminal group R 10 wherein R 10 is hydrogen or a group that can be eliminated when reacting with the payload group each L 1 is independently a cleavable sequence 1 containing an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1 to 10 amino acids each L 2 is independently a bond or C 2-20 alkylene, and one or more -CH2- structures in the alkylene are -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, TIFF2025524725000014.tif12165C 4-10 cycloalkylene, C 4-10 heterocyclylene, phenylene may be replaced, wherein the cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R 9 are each substituted with at least one substituent selected from Ld2 and each Ld1 are independently a bond or -NH-C 1-20Alkylene-(CO)-, -NH-(PEG) i Selected from -(CO)-, or independently, unsubstituted or having a side chain of -(PEG) j -R 11 Is a natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10 and substituted with -(PEG) t -, -(PEG) i - and -(PEG) j - is a PEG fragment containing the indicated number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, with an optional additional C at one end 1-10 Having alkylene R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 Are each independently hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 Selected from cycloalkylene, or R 1 And R 2 Together with the carbon atom to which they are attached, form a 3- to 6-membered cycloalkyl group, or R 3 And R 4 Together with the carbon atom to which they are attached, form a 3- to 6-membered cycloalkyl group, and R 11 Is C 1-10 Alkyl, m is any integer from 1 to 3, n is any integer from 2 to 20, d is 0 or any integer from 1 to 6, Each i is independently an integer from 0 to 100, preferably from 0 to 20, preferably each i is independently an integer from 0 to 12, more preferably from 0 to 8, especially 4, Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20, and preferably each j is independently an integer from 1 to 12, more preferably an integer from 8 to 12, particularly 8 or 12. Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20, and preferably each t is independently an integer from 1 to 12, more preferably an integer from 8 to 12, particularly 8 or 12.

[0042] In one embodiment, L 2 is -(CH2) p -(CH2)2(CO)-(where p is 0 or an integer from 1 to 5) selected from TIFF2025524725000015.tif82165 (where b is an integer from 1 to 10).

[0043] In one embodiment, p is from 0 to 3, preferably 3.

[0044] In one embodiment, L 2 is selected from -(C2H4-O) p -(CH2)2(CO)-, where p is an integer from 1 to 5, and more preferably p is 2 or 4.

[0045] In one embodiment, the carbonyl group in each of the above structures of L 2 is bonded to L 1 and the other bonding site is bonded to opSu.

[0046] In one embodiment, the carbonyl group in each of the above structures of L 2 is bonded to L 1 and the other bonding site is bonded to an amide. Ld2 and each Ld1 are independently a bond or TIFF2025524725000016.tif74165, each i is independently an integer from 0 to 100, each j and k are independently integers from 1 to 100.

[0047] In one embodiment, each i is independently an integer from 0 to 20. In one embodiment, each i is independently an integer from 0 to 12.

[0048] In one embodiment, each j and k are independently integers from 1 to 20. In one embodiment, each j and k are independently integers from 1 to 12.

[0049] In one embodiment, each i is independently an integer from 0 to 8, particularly 4.

[0050] In one embodiment, each j is independently an integer from 8 to 12, particularly 8 or 12.

[0051] In one embodiment, each k is independently an integer from 1 to 7, particularly 1 or 3 or 5.

[0052] In one embodiment, Ld2 and each Ld1 are independently a bond, or a C 1-20 alkylene having amino and carbonyl at each of the two ends, or a PEG fragment of a specific length having amino and carbonyl at each of the two ends (-(PEG) i - as shown), or one or more natural amino acids that are independently unsubstituted or substituted with a PEG fragment of a specific length having side chains (-(PEG) j - as shown).

[0053] In one embodiment, -(PEG) i - is -(O-C2H4) i - or -(C2H4-O) i - and an optional additional C 1-10 alkylene at one end, and -(PEG) j - is -(O-C2H4) j - or -(C2H4-O) j - and an optional additional C 1-10 alkylene at one end. In one embodiment, -(PEG) i - is -C2H4-(O-C2H4) i-or-(C2H4-O) i -contains -C2H4-.

[0054] When there are two or more Ld1, B, L in the molecule 2 , or L 1 structures, it should be understood that the structures of each Ld1, B, L 2 , or L 1 are independently selected. When there are two or more R x (where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) in the molecule, each R x is independently selected. In some embodiments, the "x" of the molecule may or may not be accompanied by an additional apostrophe (') or multiple apostrophes (e.g., '', ''', '''', etc.), for example, R, R 1’ , R 1’’ , R 1’’’ , R 2’ , R 2’’ , R 2’’’ etc., are shown, and in the formula, each R x is independently selected with or without an additional apostrophe or multiple apostrophes. Other R x , for example, R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , as well as "Ld1", "B", "L 2 ", and "L 1 " should be understood in the same way. In some embodiments, the "i" of the molecule may or may not be accompanied by an additional number, for example, i1, i2, i3, i4, etc., and the numbers do not indicate any sequence but are simply used to identify "i". Also, each "i" is independently selected with or without an additional number.

[0055] In one embodiment, the cleavable sequence 1 is selected from Gly-Gly-Phe-Gly, Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Ala-Ala-Ala, and combinations thereof, and preferably, the cleavable sequence 1 is Gly-Gly-Phe-Gly.

[0056] In one embodiment, W is hydrogen.

[0057] In one embodiment, W is -C2H4-(PEG) t -(CO)NH2, wherein t is independently an integer from 1 to 100, preferably from 1 to 20, and preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12.

[0058] In one embodiment, R 11 is C 1-6 alkyl, preferably methyl.

[0059] In one embodiment, n is an integer from 2 to 5, particularly 3.

[0060] In one embodiment, d is 0 or any integer from 1 to 4, preferably 0, 1, 2, or 3.

[0061] Thiosuccinimide is unstable under physiological conditions and prone to retro-Michael addition leading to cleavage at the conjugation site. Further, when another thiol compound is present in the system, thiol exchange between thiosuccinimide and the other thiol compound may also occur. All these reactions cause a decrease in the payload and result in toxic side effects. In the present disclosure, when applied to the linker, the ring-opening succinimide structure no longer undergoes retro-Michael addition or thiol exchange, and thus the product is more stable. The method for the ring-opening reaction can be found in WO2015165413A1.

[0062] A compound containing an open - ring succinimide moiety can be purified by semi - preparative / preparative HPLC or other suitable separation means regardless of the efficiency of the succinimide ring - opening reaction to obtain a composition of defined high purity.

[0063] A moiety containing a recognition sequence of a ligase receptor or donor substrate In one embodiment, the G of the compound of formula (I) n moiety is a recognition sequence of a ligase receptor substrate, whereby the enzyme - catalyzed coupling of the compound of formula (I) and a targeting molecule under the catalysis of a ligase is promoted. The targeting molecule is optionally modified and contains the corresponding recognition sequence of the ligase receptor substrate.

[0064] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of natural transpeptidases, non - natural transpeptidases, their variants, and combinations thereof. Non - natural transpeptidase enzymes can be obtained by manipulation of natural transpeptidases, but are not limited thereto. In a preferred embodiment, the ligase is selected from the group consisting of natural sortases, non - natural sortases, and combinations thereof. Species of natural sortases include sortase A, sortase B, sortase C, sortase D, L. plantarum sortase, etc. (detailed descriptions can be found in US20110321183A1, which is incorporated herein by reference). The type of ligase corresponds to the ligase recognition sequence and is thereby used to achieve specific conjugation between different molecules or structural fragments.

[0065] In some embodiments, the ligase is a sortase selected from sortase A, sortase B, sortase C, sortase D, and L. plantarum sortase. In these embodiments, the recognition sequence of the ligase acceptor substrate is selected from the group consisting of oligomeric glycine, oligomeric alanine, and a mixture of oligomeric glycine / alanine having a degree of polymerization of 3 to 10. In certain embodiments, the recognition sequence of the ligase acceptor substrate is G n where G is glycine (Gly) and n is an integer from 2 to 10.

[0066] In another particular embodiment, the ligase is sortase A from Staphylococcus aureus. Thus, the ligase recognition sequence can be the normal recognition sequence of the enzyme as LPXTG. In yet another particular embodiment, the recognition sequence of the ligase donor substrate is LPXTGJ, and the recognition sequence of the ligase acceptor substrate is G n where X can be any natural or non-natural single amino acid, and J is an optionally labeled amino acid fragment that is absent or contains 1 to 10 amino acids. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment containing 1 to 10 amino acids, and each amino acid is independently any natural or non-natural amino acid. In another embodiment, J is G m where m is an integer from 1 to 10. In yet another particular embodiment, the recognition sequence of the ligase donor substrate is LPETG. In another particular embodiment, the recognition sequence of the ligase donor substrate is LPETGG.

[0067] In one embodiment, the ligase is sortase B from Staphylococcus aureus, and the corresponding donor substrate recognition sequence can be NPQTN. In another embodiment, the ligase is sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence can be NPKTG.

[0068] In yet another embodiment, the ligase is sortase A from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, where J is as defined above. In another embodiment, the ligase is from the sortase subfamily 5 of Streptomyces coelicolor, and the corresponding donor substrate recognition sequence may be LAXTG.

[0069] In yet another embodiment, the ligase is sortase A from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ.

[0070] The ligase recognition sequence can also be any other completely novel recognition sequence for transpeptidase optimized by manual screening.

[0071] The moiety containing the reactive group The reactive group for binding to the payload In one embodiment, B is the terminal group R 10 and L 1The cleavable array 1 in [context] binds to the payload. In such cases, B is not present within the molecule obtained as a result of the binding of the cleavable array 1 and the payload. In one embodiment, B is used for binding to the payload. With respect to binding to the payload, the compound of formula (I) contains a reactive group. In one embodiment, B in the compound of formula (I) is bound to the payload via an amide bond or an ester bond or an ether bond. In one embodiment, the reactive groups of B in formula (I) are independently reactive groups for condensation reactions, nucleophilic addition or electrophilic addition (e.g., reactive C=O moieties, reactive C=C-C=O moieties, amino groups, amine groups, hydroxy groups or thiol groups), or reactive groups for substitution reactions (e.g., leaving groups attached to O, C, N, or S atoms). In one embodiment, the reactive groups of B are independently selected from carboxyl groups, active esters, aldehyde groups, amino groups, amine groups, hydroxy groups, and thiol groups. In a specific embodiment, the reactive groups of B used for binding to the payload are independently selected from amino groups, amine groups, hydroxy groups, thiol groups, carboxyl groups, and active esters.

[0072] In one embodiment, the reactive groups of B are independently amino groups, amine groups, or hydroxy groups, which react with the corresponding groups of the payload (e.g., carboxyl groups, sulfonic acid groups, phosphoryl groups having a free - OH terminus, active esters, acid chlorides, or isocyanate groups). In another embodiment, the reactive groups of B are independently carboxyl groups or active esters, which react with the corresponding groups of the payload (e.g., amino groups, amine groups or hydroxy groups).

[0073] In one embodiment, the reactive groups of B are independently amino groups, hydroxy groups or thiol groups, which react with the corresponding groups of the payload (e.g., halogens, hydroxy groups, aldehyde groups). In another embodiment, the reactive group of B is independently a hydroxy group, which reacts with the corresponding group of the payload (e.g., halogen or hydroxy group).

[0074] In one embodiment, each B is independently R 10 or the following (1), (2), and (3): (1) a self-destructive spacer Sp1, (2) a bond, or -CR 1 R 2 -, C 1-10 an alkylene, and one or a combination of two or more of the divalent groups selected from -(CO)-, and (3) a terminal group R 10 is a combination of

[0075] In one embodiment, Sp1 is selected from PABC, acetal, heteroacetal, and combinations thereof. In one embodiment, Sp1 is an acetal, heteroacetal, or PABC. In one embodiment, the heteroacetal is selected from N,O-heteroacetals. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is bonded to the cleavable sequence 1, and U is absent or is O, S, or NH, preferably O or S. In one embodiment, U is absent or is O, S, or NH, preferably O or S.

[0076] In one embodiment, B is R 10 , -NH-CH2-U-R 10 , -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 or -NH-CH2-U-(CH2) g -(CO)-R 10 is

[0077] In one embodiment, R 10 is hydrogen, hydroxy, or TIFF2025524725000017.tif26165. In one embodiment, R 10 is hydrogen. In one embodiment, R 10 is hydroxy or TIFF2025524725000018.tif is 32165.

[0078] In one embodiment, R 10 represents a portion of the structure that will not appear in the product molecule resulting from the reaction of B with the payload.

[0079] Specific embodiments of the compound of formula (I) In one embodiment, W is hydrogen, and each LKa is TIFF2025524725000019.tif is 27165. In one embodiment, formula (I) has the structure of formula (I-a) TIFF2025524725000020.tif has the structure of 47165.

[0080] In one embodiment, each LKa is TIFF2025524725000021.tif is 27165. In one embodiment, formula (I) has the structure of formula (I-b) TIFF2025524725000022.tif has the structure of 58165.

[0081] In one embodiment, Ld2 is a bond and d is 0. In one embodiment, the compound of formula (I-a) is as follows. TIFF2025524725000023.tif 27165

[0082] In one embodiment, d is 0 and Ld2 is TIFF2025524725000024.tif is 27165. In one embodiment, the compound of formula (I-a) is as follows. TIFF2025524725000025.tif 32165

[0083] In one embodiment, d is 1, 2, or 3, and Ld2 and each Ld1 are independently selected from TIFF2025524725000026.tif 27165. In one embodiment, the compound of formula (I-a) is as follows. TIFF2025524725000027.tif143165

[0084] In one embodiment, Ld2 is TIFF2025524725000028.tif43165, and d is 0. In one embodiment, the compound of formula (I-a) is as follows. TIFF2025524725000029.tif47165

[0085] In one embodiment, d is 1, 2, or 3, and Ld2 is TIFF2025524725000030.tif45165, and each Ld1 is independently selected from TIFF2025524725000031.tif26165. In one embodiment, the compound of formula (I-a) is as follows. TIFF2025524725000032.tif200165

[0086] In one embodiment, d is 1, W is hydrogen, and Ld2 is TIFF2025524725000033.tif42165, and each Ld1 is independently selected from TIFF2025524725000034.tif27165. In one embodiment, the compound of formula (I-b) is as follows. TIFF2025524725000035.tif79165

[0087] In one embodiment, d is 1, and W is -C2H4-(PEG) t -C(O)NH2, Ld2 is a bond, and each Ld1 is independently selected from TIFF2025524725000036.tif27165. In one embodiment, the compound of formula (I-b) is as follows. TIFF2025524725000037.tif63165

[0088] In one embodiment, n is 3, and L 2 is -(CH2) p -(CH2)2(CO)-, p is 3, and L 1 is GGFG, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 and U is O, and g is 1. In one embodiment, I-a-0-1 has the structure: TIFF2025524725000038.tif92165.

[0089] In one embodiment, I-a-0-2 has the structure: TIFF2025524725000039.tif78165.

[0090] In one embodiment, I-a-0-3 has the structure: TIFF2025524725000040.tif141165.

[0091] In one embodiment, I-a-0-4 has the structure: TIFF2025524725000041.tif177165TIFF2025524725000042.tif104165.

[0092] In one embodiment, I-a-0-5 has the structure: TIFF2025524725000043.tif171165TIFF2025524725000044.tif93165.

[0093] In one embodiment, I-a-1-1 has the structure: TIFF2025524725000045.tif136165.

[0094] In one embodiment, I-a-1-2 has the structure: It has TIFF2025524725000046.tif167165.

[0095] In one embodiment, I-a-1-3 has the structure: It has TIFF2025524725000047.tif130165 TIFF2025524725000048.tif122165 TIFF2025524725000049.tif134165.

[0096] In one embodiment, I-a-1-4 has the structure: It has TIFF2025524725000050.tif202165 TIFF2025524725000051.tif113165.

[0097] In one embodiment, n is 3, and L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, and L 1 is GGFG, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 and U is O, g is 1. In one embodiment, I-b-1 has the structure: It has TIFF2025524725000052.tif177165 TIFF2025524725000053.tif86165.

[0098] In one embodiment, n is 3, and L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, and L 1 is GGFG, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10where U is O and g is 1. In one embodiment, I-b-0 has the structure: It has TIFF2025524725000054.tif205165.

[0099] In one embodiment, i is 4, g is 1, and R 11 is methyl.

[0100] In one embodiment, i is 2, g is 1, and R 11 is methyl.

[0101] In one embodiment, I-a-0-2 is as follows (I-a-0-2-1 to I-a-0-2-3). TIFF2025524725000055.tif98165

[0102] In one embodiment, i is 4, j is 8, g is 1, and R 11 is methyl. In one embodiment, I-a-1-2 is as follows (I-a-1-2-1 to I-a-1-2-3). TIFF2025524725000056.tif190165

[0103] In one embodiment, n is 3, i is 4, j is 12, g is 1, and R 11 is methyl. In one embodiment, I-a-1-2 is as follows (I-a-1-2-4 to I-a-1-2-6). TIFF2025524725000057.tif194165

[0104] In one embodiment, i is 4, j is 8, g is 1, m is 1, n is 3, and R 11 is methyl. In one embodiment, I-b-1 is as follows (I-b-1-1 to I-b-1-3). TIFF2025524725000058.tif193165TIFF2025524725000059.tif94165

[0105] In one embodiment, i is 4, j is 12, g is 1, m is 1, n is 3, and R 11 is methyl. In one embodiment, I-b-1 is as follows (I-b-1-4 to I-b-1-1-6). TIFF2025524725000060.tif197165TIFF2025524725000061.tif99165

[0106] In one embodiment, i is 4, j is 8, g is 1, m is 2, n is 3, and R 11 is methyl. In one embodiment, I-b-1 is as follows (I-b-1-7 to I-b-1-9). TIFF2025524725000062.tif208165TIFF2025524725000063.tif101165

[0107] In one embodiment, i is 4, j is 12, g is 1, m is 2, n is 3, and R 11 is methyl. In one embodiment, I-b-1 is as follows (I-b-1-10 to I-b-1-12). TIFF2025524725000064.tif200165TIFF2025524725000065.tif96165

[0108] In one embodiment, i is 4, t is 8, g is 1, m is 1, n is 3, and R 11 is methyl. In one embodiment, I-b-0 is as follows (I-b-0-1 to I-b-0-3). TIFF2025524725000066.tif224165

[0109] In one embodiment, i is 4, t is 12, g is 1, m is 1, n is 3, and R 11 is methyl. In one embodiment, I-b-0 is as follows (I-b-0-4 to I-b-0-6). TIFF2025524725000067.tif152165TIFF2025524725000068.tif81165

[0110] In one embodiment, i is 4, t is 8, g is 1, m is 2, n is 3, and R 11 is methyl. In one embodiment, I-b-0 is as follows (I-b-0-7 to I-b-0-9). TIFF2025524725000069.tif157165TIFF2025524725000070.tif76165

[0111] In one embodiment, i is 4, t is 12, g is 1, m is 2, n is 3, and R 11 is methyl. In one embodiment, I-b-0 is as follows (I-b-0-10 to I-b-0-12). TIFF2025524725000071.tif157165TIFF2025524725000072.tif81165

[0112] A compound of formula (I) having a payload The reactive group contained by B is covalently conjugated with a payload containing another reactive group to obtain a compound of formula (I) having a payload.

[0113] In yet another aspect, formula (II) A compound having the structure of TIFF2025524725000073.tif32165 is provided, wherein Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P, M is hydrogen or LKa-LKb-P, provided that Q and M are not both hydrogen at the same time, P is a payload that binds to the B moiety or the L 1 moiety of the compound of formula (I), n, d, Ld1, Ld2, t, LKa, and LKb are as defined in formula (I).

[0114] As defined above in the present specification, in the compound of formula (I), each LKb is independently L 2 -L 1 -B, and each B is independently a terminal group R 10 or one of the following (1), (2), and (3): (1) a self-destructive spacer Sp1, (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and one or a combination of two or more of the divalent groups selected from -(CO)-, and (3) a combination of the terminal group R 10 wherein R 10 is hydrogen or a group that can be eliminated when reacting with the payload group. In one embodiment, R 10 represents a portion of a structure that will not appear within the product molecule resulting from the reaction between B and the payload.

[0115] In one embodiment, P is attached to the B portion of the compound of formula (I) to form a compound of formula (II). As defined above, R 10 does not appear in the B-P structure of the compound of formula (II).

[0116] When B of the compound of formula (I) is the terminal group R 10 , it should be understood that R 10 will not appear in the compound of formula (II). Thus, as a result, B does not exist in the B-P structure of the compound of formula (II).

[0117] [[ID=)]]In one embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P, wherein P is a payload attached to the B portion or the L 1 portion of the compound of formula (I), and each B is independently a terminal group R10 or the following (1), (2), and (3): (1) a self-destructive spacer Sp1, (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and one or a combination of two or more of the divalent groups selected from -(CO)-, and (3) a terminal group R 10 is a combination of, R 10 is hydrogen or a group that can be eliminated when reacting with the payload group, R 10 represents a portion of a structure that will not appear in the product molecule resulting from the reaction between B and the payload.

[0118] In one embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P, wherein each B independently does not exist or is the following (1) and (2): (1) a self-destructive spacer Sp1, and (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and one or a combination of two or more of the divalent groups selected from -(CO)- is a combination of. In a preferred embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P, wherein each B independently does not exist or is -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-. In another embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P. In one embodiment, in LKa-L 2 -L 1 -B-P, B does not exist. In one embodiment, in LKa-L 2-L 1 -B-P, where B is the following (1) and (2): (1) a self-destructive spacer Sp1, and (2) a combination of one or more of the divalent groups selected from bonding, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and a combination of one or more of the divalent groups selected from -(CO)- is a combination of. In one embodiment, LKa-L 2 -L 1 -B-P, where B is -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, and U is absent or is O, S or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is bonded to the payload via an amide bond or an ester bond or an ether bond.

[0119] As defined above herein, when B of the compound of formula (I) is the terminal group R 10 , B is not present in the B-P structure of the compound of formula (II). In such a case, the cleavable sequence 1 in L 1 binds to the payload to form the compound of formula (II), and it can also be understood that B is not present in the molecule formed by the binding of the cleavable sequence 1 and the payload. Therefore, in one embodiment, P binds to the L 1 portion of the compound of formula (I) to form the compound of formula (II). Therefore, in one embodiment, M is LKa-L 2 -L 1 -B-P, where B is absent, and M can also be represented as LKa-L 2 -L 1 -P.

[0120] Payload In the present disclosure, the payload can be selected from the group consisting of small molecule compounds, nucleic acids and analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics, and proteins. In one embodiment, the payload is selected from the group consisting of small molecule compounds, nucleic acid molecules, and tracer molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from the group consisting of cytotoxins and fragments thereof.

[0121] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is a taxane, a mitansine, an auristatin, an epothilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, an indole-sulfonamide, a vincblastine, such as vincblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin 10 and analogs, halichondrin B and eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, and is selected from the group consisting of. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, such as camptothecin and its derivatives, mitoxantrone, mitoguazone. In a preferred embodiment, the cytotoxin is a nitrogen mustard, such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenamet, phenesterine, prednimustine, trofosfamide, uracil mustard, and is selected from the group consisting of. In yet another preferred embodiment, the cytotoxin is a nitrosourea, such as carmustine, flubenzuron, formoterol, lomustine, nimustine, ranimustine, and is selected from the group consisting of. In one embodiment, the cytotoxin is selected from the group consisting of aziridines. In a preferred embodiment, the cytotoxin is selected from the group consisting of bendopar, carbocon, meturedepa, and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, and aclacinomycin.In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycin, actinomycin C, carabicin, calminomycin, and cardinophilin, calminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferric adriamycin, rhodrubicin, lufocromomycin, streptozocin, dinostatin, zorubicin. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, baculocpolin A, and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of folic acid dimethyl, methotrexate, pteropterin, trimethoprim, and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamiprine, thioguanine. In yet another embodiment, the cytotoxin is selected from pyrimidine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone. In another embodiment, the cytotoxin is selected from the group consisting of antiadrenal drugs.In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of antiandrogens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate, and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors. In another embodiment, the cytotoxin is selected from the group consisting of vinblastine, colchicine, taxane, auristatin, maytansinoid, calicheamicin, doxorubicin, duocarmucin, SN-38, cryptophycin analog, deruxtecan, duocarmazine, calicheamicin, centanamycin, drastansine, and pyrrolobenzodiazepine (PBD). In a particular embodiment, the cytotoxin is selected from the group consisting of vinblastine, colchicine, taxane, auristatin, and maytansinoid.

[0122] In a particular embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f, etc.

[0123] In another particular embodiment, the cytotoxin is a maytansinoid, such as DM1, etc. It should be noted that when a cytotoxin containing a thiol moiety is used, the thiol moiety can react with a maleimide moiety to form a thiosuccinimide, such as a maytansinoid, such as DM1, and the cytotoxin can be directly conjugated via the thiosuccinimide. In such cases, in some embodiments, the payload and the thiol moiety together constitute the cytotoxin, and thus, in such cases, it can be understood that the payload represents the remaining portion of the cytotoxin molecule excluding the thiol moiety.

[0124] In certain embodiments, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), and the like. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0125] The payload contains a reactive group that reacts with the reactive group in the compound of formula (I) and can thus covalently conjugate the payload to the compound of formula (I). Compounds that do not contain a reactive group require appropriate derivatization to obtain the payload.

[0126] In one embodiment, the cytotoxin is a compound of the following formula (i) TIFF2025524725000074.tif68165, wherein a * is 0 or 1, p1 * and p2 * the carbon atoms marked with are each an asymmetric center, and the asymmetric centers are in the S configuration, R configuration, or racemic form, L 1* is unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH2, C 1-6 selected from alkylene, M * is -CH2-, -NH-, or -O-, L 2* is C 1-3 alkylene, R 1* and R 2* are each independently selected from hydrogen, C 1-6 alkyl, halogen, and C 1-6 alkoxy.

[0127] In certain embodiments, the cytotoxin is a compound of the following formula (i') TIFF2025524725000075.tif50165, In the formula, g is an arbitrary integer from 1 to 6.

[0128] In one embodiment, g * is an arbitrary integer from 1 to 3, preferably 1.

[0129] In one embodiment, L 1* is C 1-6 linear alkylene, C 1-6 branched alkylene, C 3-6 cyclic alkylene and C 3-4 cyclic alkyl-C 1-2 selected from linear alkylene groups, each of which is independently unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH2. In one embodiment, L 1* is selected from unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH2, C 1-4 alkylene. In a preferred embodiment, L 1* is -CH2-, -C2H4-, TIFF2025524725000076.tif24165 selected from, each of which is independently unsubstituted or substituted with at least one substituent selected from halogen, -OH, and -NH2. In a preferred embodiment, L 1* is -CH2-, TIFF2025524725000077.tif25165 selected from, where "#" indicates the position bonded to the carbonyl. In a more preferred embodiment, L 1* is -CH2-, TIFF2025524725000078.tif22165 selected from, where "#" indicates the position bonded to the carbonyl. In a particular embodiment, L 1* is -CH2-, TIFF2025524725000079.tif22165 selected from, where "#" indicates the position bonded to the carbonyl. In a preferred embodiment, the halogen is selected from F, Cl, and Br, particularly F.

[0130] In one embodiment, a * is 1, M * is -CH2-, -NH-, or -O-, and L 2* is -C2H4-. In another embodiment, a * is 1, M * is -CH2-, and L 2* is -CH2-. In one embodiment, a * is 0.

[0131] In one embodiment, the carbon atom marked with p1 * is in the S configuration or a racemate, preferably the S configuration. In another embodiment, the carbon atom marked with p2 * is in the S configuration or a racemate, preferably the S configuration.

[0132] In one embodiment, R 1* and R 2* are each independently selected from hydrogen, C 1-3 alkyl, halogen, and C 1-3 alkoxy. In a preferred embodiment, R 1* and R 2* are each independently selected from CH3-, F, Cl, Br, and CH3O-. In one embodiment, R 1* is selected from CH3- and Cl. In another embodiment, R 2* is F.

[0133] In one embodiment, a * is 0, and L 1* is selected from -CH2-, TIFF2025524725000080.tif22165, where "#" indicates the position bonded to the carbonyl. In one embodiment, a * is 1, and L 1* is TIFF2025524725000081.tif22165, M * is O, and L 2* is -C2H4-.

[0134] In one embodiment, a * is 0, R 1* is Cl, R 2* is F, and L 1* is selected from -CH2-, TIFF2025524725000082.tif22165. In one embodiment, a * is 0, R 1* is CH3-, R 2* is F, and L 1* is selected from TIFF2025524725000083.tif22165, where " #" indicates the position bonded to the carbonyl.

[0135] In one embodiment, a * is 1, R 1* is CH 3- and R 2* is F, and L 1* is TIFF2025524725000084.tif19165, M is O, and L 2* is -C2H4-.

[0136] In one embodiment, the cytotoxin is selected from the following compounds, where the wavy bond indicates the binding site for binding to the compound of formula (I). TIFF2025524725000085.tif217165

[0137] In some embodiments, the payload is DX8951f (compound 9), DXd-(1) (compound 10), DXd-(2) (compound 14), TIFF2025524725000086.tif47165 Preferably, DX8951f, DXd-(1) TIFF2025524725000087.tif47165 More preferably DXd-(1), TIFF2025524725000088.tif45165 Most preferably, TIFF2025524725000089.tif45165 is selected from.

[0138] Preparation of Compounds of Formula (I) with a Payload In one embodiment, the linking unit and the payload are linked via a reactive group as defined above using any reaction known in the art including, but not limited to, condensation reactions, nucleophilic additions, electrophilic additions, etc.

[0139] In one embodiment, the payload is a cytotoxin. In one embodiment, the linker-payload intermediate (numbered as LBx) is as shown in the following table. TIFF2025524725000090.tif223165TIFF2025524725000091.tif54165

[0140] Conjugates and Their Preparation Furthermore, compounds of Formula (I) having a payload with a portion containing a ligase recognition sequence can conjugate with other molecules containing the ligase recognition sequence and thereby can be used, for example, in the preparation of targeted molecule-drug conjugates. Accordingly, in yet another aspect, conjugates comprising a compound of Formula (I), a targeting molecule, and a payload are provided.

[0141] In yet another aspect, Formula (III): Conjugates having the structure of TIFF2025524725000092.tif35165 are provided, wherein n, d, Ld1 and Ld2 are as defined in Formula (I), Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb―P, M is hydrogen or LKa-LKb―P, provided that Q and M are not both hydrogen at the same time, P is a payload that binds to the B moiety or the L 1 moiety of the compound of Formula (I), A is the G of the compound of Formula (I) nAn anti-Trop2 antibody or an antigen-binding fragment thereof that binds to the moiety, where G is glycine, z is an integer from 1 to 20.

[0142] In one embodiment, LKa and LKb are as defined in formula (I).

[0143] In one embodiment, the conjugate has a drug-to-antibody ratio (DAR) that is an integer or non-integer from 1 to 20.

[0144] As defined above herein, in one embodiment, G of the compound of formula (I) n The moiety is a recognition sequence of a ligase receptor substrate, thereby facilitating the enzyme-catalyzed coupling of the compound of formula (I) and the targeting molecule under the catalytic action of the ligase. The targeting molecule is optionally modified and contains the corresponding recognition sequence of the ligase receptor or donor substrate.

[0145] When the antibody (or antigen-binding fragment) conjugates with the G n It should be understood that the recognition sequence of the ligase receptor substrate and the recognition sequence of the ligase donor substrate react with each other to form the resulting sequence.

[0146] In one embodiment, the antibody (or antigen-binding fragment) contains LPXTGJ as the recognition sequence of the ligase donor substrate, and J is as defined above. G, which is the corresponding recognition sequence of the ligase receptor substrate n When conjugated with, the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate binds to the free N-terminus of G n A new peptide bond is formed. The resulting sequence is LPXTG n which is. The sequences G n and LPXTGJ are as defined above.

[0147] In one embodiment, P is the B moiety or L of the compound of formula (I) 1is attached to the moiety, and A is the G of the compound of formula (I) n is attached to the moiety, and a compound of formula (III) is formed.

[0148] As defined above, R 10 does not appear in the B-P structure of the compound of formula (III). As defined above, when B of the compound of formula (I) is the terminal group R 10 it is not present in the B-P structure of the compound of formula (III).

[0149] As defined above, in the A-G n structure of the compound of formula (III), A optionally contains the corresponding sequence resulting from the reaction of the recognition sequence of the ligase acceptor substrate and the recognition sequence of the ligase donor substrate.

[0150] In one embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P, wherein P is a payload that binds to the B moiety or the L 1 moiety of the compound of formula (I), each B is independently the terminal group R 10 or one or more combinations of the following (1), (2), and (3): (1) a self-destructive spacer Sp1, (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and -(CO)-, and (3) a combination of the terminal group R 10 wherein R 10 is hydrogen or a group that can be eliminated when reacting with the group of the payload, and R 10 represents a part of the structure that will not appear in the product molecule resulting from the reaction of B and the payload.

[0151] In one embodiment, M is hydrogen or LKa-L 2 -L 1-B-P, wherein each B is independently non-existent or is one or a combination of two or more of the following (1) and (2): (1) a self-destructive spacer Sp1, and (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and one or a combination of two or more of the divalent groups selected from -(CO)- is a combination. In a preferred embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P, wherein each B is independently non-existent or is -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-. In another embodiment, M is hydrogen or LKa-L 2 -L 1 -B-P. In one embodiment, in LKa-L 2 -L 1 -B-P, B is non-existent. In one embodiment, in LKa-L 2 -L 1 -B-P, B is the following (1) and (2): (1) a self-destructive spacer Sp1, and (2) a bond, or -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and one or a combination of two or more of the divalent groups selected from -(CO)- is a combination. In one embodiment, in LKa-L 2 -L 1 -B-P, B is -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g-(CO)-, and U is absent or is O, S or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is linked to the payload via an amide bond or an ester bond or an ether bond. In one embodiment, M is LKa-L 2 -L 1 -B-P, where B is absent and M can also be represented as LKa-L 2 -L 1 -P.

[0152] Targeting molecule In one embodiment, the targeting molecule is an anti-Trop2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), and V H comprises (i) HCDR1 comprising the amino acid sequence of X1X2GMX3 (SEQ ID NO: 1), where X1 is N, T or A, X2 is Y or A, and X3 is N or Q, (ii) HCDR2 comprising the amino acid sequence of WINTX4X5GX6PX7YX8X9DFKG (SEQ ID NO: 2), where X4 is Y, H or D, X5 is T or S, X6 is E or V, X7 is T or K, X8 is T or A, and X9 is D or E, (iii) HCDR3 comprising the amino acid sequence of X 10 GFGSSYWYFDV (SEQ ID NO: 3), where X 10 is G or S, and / or V L comprises (i) LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14), (ii) LCDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 15), and (iii) LCDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) .

[0153] In one embodiment, HCDR1 comprises the amino acid sequence of NYGMN, TAGMQ, AAGMN or NAGMN.

[0154] In one embodiment, HCDR2 comprises the amino acids of WINTYTGEPTYTDDFKG (SEQ ID NO: 8), WINTHSGVPKYAEDFKG (SEQ ID NO: 9), WINTDSGEPTYTDDFKG (SEQ ID NO: 10).

[0155] In one embodiment, HCDR3 comprises the amino acids of GGFGSSYWYFDV (SEQ ID NO: 11) or SGFGSSYWYFDV (SEQ ID NO: 12).

[0156] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), V H comprises (i) HCDR1 comprising the amino acid sequence of X1AGMN, where X1 is N or A, (ii) HCDR2 comprising the amino acid sequence of WINTDSGEPTYTDDFKG (SEQ ID NO: 10), (iii) HCDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 11) and / or, V L comprises (i) LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14), (ii) LCDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 15), (iii) LCDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) and includes.

[0157] In one embodiment, V H comprises (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) An HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) An HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 and / or V L is (i) An LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) An LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) An LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 and comprises.

[0158] In one embodiment, V H is (i) An HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, (ii) An HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) An HCDR3 comprising the amino acid sequence of SEQ ID NO: 12 and / or V L is (i) An LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, (ii) An LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) An LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 and comprises.

[0159] In one embodiment, V H is (i) An HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) An HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) An HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 and / or V L is (i) An LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) An LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) An LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 and comprises.

[0160] In one embodiment, V H is (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 7, (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 10, and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 11 and / or V L is (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 13, (ii) LCDR2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 16 contains.

[0161] In one embodiment, V H is (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 6, (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 8, and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 11 and / or V L is (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 13, (ii) LCDR2 containing the amino acid sequence of SEQ ID NO: 15, and (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 16 contains.

[0162] In one embodiment, V H has the structure: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4, where FR1 contains the amino acids of SEQ ID NO: 17, FR2 contains the amino acids of SEQ ID NO: 18, FR3 contains the amino acids of SEQ ID NO: 19, and FR4 contains the amino acids of SEQ ID NO: 20.

[0163] In one embodiment, V HIt includes an amino acid sequence having at least about 90% sequence identity to the amino acid sequences of SEQ ID NOs: 21 to 25. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 21. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 22. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 23. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 24. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 25.

[0164] In one embodiment, V L includes an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27. In one embodiment, V L includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V L includes the amino acid sequence of SEQ ID NO: 27.

[0165] In one embodiment, V H includes an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and / or V L includes an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

[0166] In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 21, and V L includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 22, and V L includes the amino acid sequence of SEQ ID NO: 27. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 23, and V L includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 24, and V Lincludes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V H includes the amino acid sequence of SEQ ID NO: 25, and V L includes the amino acid sequence of SEQ ID NO: 26.

[0167] In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant domain (CH) that includes an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, and / or a light chain constant domain that includes an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0168] In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant domain (CH) that includes the amino acid sequence of SEQ ID NO: 28, and a light chain constant domain that includes the amino acid sequence of SEQ ID NO: 29.

[0169] In one embodiment, the antibody or antigen-binding fragment binds to TROP2 with an equilibrium dissociation constant (K D ) of about 0.5 nM to about 20 nM. In one embodiment, the value of K D is about 0.5 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 15 nM, about 18 nM, about 20 nM, and / or a range between any two values (including the values at both ends). In one embodiment, the value of K<## D is about 7.5 nM to about 13.5 nM.

[0170] In one embodiment, the targeting molecule is an anti-human TROP2 antibody or an antigen-binding fragment thereof.

[0171] In one embodiment, the antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimetics. In one embodiment, the antibody mimetic is selected from scFv, minibody, diabody, and nanobody.

[0172] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequences of SEQ ID NOs: 30-33, and / or a light chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35.

[0173] At least about 90% is about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or a range between any two values (including the end values).

[0174] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NOs: 30-33, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35.

[0175] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0176] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0177] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0178] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0179] Examples of anti-human TROP2 antibodies include, but are not limited to, the antibody of Trodelvy (hRS7) and the antibody of DS1062 (datopotamab). The sequences of the antibodies are shown in Table 1.

[0180] In one embodiment, the pharmaceutical composition comprises an antibody or an antigen-binding fragment.

[0181]

Table 1

[0182] Antibody conjugated with a linker-payload With respect to conjugation with the compound of formula (I), the antibody of the present disclosure may comprise a modification moiety for binding to Gn of the compound of formula (I). The introduction position of such a modification moiety is not limited, and for example, the introduction position may be located at the C-terminus or N-terminus of the heavy chain or light chain of the antibody, but is not limited thereto.

[0183] In one embodiment, the conjugate of the present disclosure formed by conjugation of an anti-human TROP2 antibody and a payload can specifically bind to TROP2 on the surface of tumor cells and selectively kill TROP2-expressing tumor cells. In another preferred embodiment, there is provided the use of the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, disorder or condition selected from TROP2-positive tumors. In a more preferred embodiment, the disease, disorder or condition is selected from breast cancer, urothelial cancer, lung cancer, liver cancer, endometrial cancer, head and neck cancer, ovarian cancer, etc.

[0184] In an alternative embodiment, the modification moiety for conjugation with Gn in the compound of formula (I) can be introduced at a non-terminal position of the heavy chain or light chain of the antibody, for example, using a chemical modification method.

[0185] In one embodiment, the targeted molecule of the present disclosure is an antibody or an antigen-binding fragment thereof that may include a terminal modification. The terminal modification refers to a modification at the C-terminus or N-terminus of the heavy chain or light chain of the antibody, which includes, for example, a ligase recognition sequence. In another embodiment, the terminal modification may further include a spacer Sp2 containing 2 to 100 amino acids, and the antibody, Sp2, and ligase recognition sequence are sequentially linked. In a preferred embodiment, Sp2 is a spacer sequence containing 2 to 20 amino acids. In a specific embodiment, Sp2 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, particularly GA.

[0186] In a preferred embodiment, the light chain of the antibody or its antigen-binding fragment includes three types: wild-type (LC), C-terminal modified light chain (LCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and C-terminal modified light chain (LCCT L ) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. The heavy chain of the antibody or its antigen-binding fragment includes three types: wild-type (HC), C-terminal modified heavy chain (HCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and C-terminal modified heavy chain (HCCT L ) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. X can be any natural or unnatural single amino acid. In one embodiment, X is glycine. The sequences of the modified antibodies are shown in Table 2.

[0187] The conjugate of the present disclosure may further include a payload. The payload is as described above.

[0188] In one embodiment, the pharmaceutical composition includes an antibody-drug conjugate (ADC).

[0189]

Table 2

[0190] Specific embodiments of the conjugate In one embodiment, Q is hydrogen and each LKa is TIFF2025524725000099.tif25165. In one embodiment, formula (III) is formula (III-a): TIFF2025524725000100.tif58165 and has the structure of wherein A is an anti-TROP2 antibody or an antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n

[0191] In one embodiment, Ld2 is a bond and d is 0. In one embodiment, the compound of formula (III-a) is as follows. TIFF2025524725000101.tif38165

[0192] In one embodiment, d is 0 and Ld2 is TIFF2025524725000102.tif25165. In one embodiment, the compound of formula (III-a) is as follows. TIFF2025524725000103.tif36165

[0193] In one embodiment, d is 1, 2 or 3 and Ld2 and each Ld1 are independently selected from TIFF2025524725000104.tif24,165. In one embodiment, the compound of formula (III-a) is as follows. TIFF2025524725000105.tif154165

[0194] In one embodiment, Ld2 is TIFF2025524725000106.tif45165 and d is 0. In one embodiment, the compound of formula (III-a) is as follows. TIFF2025524725000107.tif59165

[0195] In one embodiment, d is 1, 2, or 3, and Ld2 is TIFF2025524725000108.tif47165, and each Ld1 is independently selected from TIFF2025524725000109.tif27165. In one embodiment, the compound of formula (III-a) is as follows. TIFF2025524725000110.tif211165

[0196] In one embodiment, z is from 1 to 4. In one embodiment, z is 2 or 4. In one embodiment, z is 2. In one embodiment, in conjugates III-a-0-1, III-a-0-2, III-a-1-1, z is 2 or 4. In one embodiment, in conjugates III-a-0-3, III-a-0-4, III-a-0-5, III-a-1-3, and III-a-1-4, z is 2. In one embodiment, in conjugate III-a-1-2, z is 4.

[0197] In one embodiment, LKa is TIFF2025524725000111.tif27165. In one embodiment, formula (III) is formula (III-b): having the structure of TIFF2025524725000112.tif74165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as such.

[0198] In one embodiment, Q is hydrogen, d is 1, and Ld2 is TIFF2025524725000113.tif43165, and Ld1 is selected from TIFF2025524725000114.tif27165. In one embodiment, the compound of formula (III-b) is as follows. TIFF2025524725000115.tif84165

[0199] In one embodiment, Q is -C2H4-(PEG) t -(CO)NH2, d is 1, Ld2 is a bond, and Ld1 is selected from TIFF2025524725000116.tif27165. In one embodiment, the compound of formula (III-b) is as follows. TIFF2025524725000117.tif64165

[0200] In one embodiment, z is from 1 to 4. In one embodiment, z is 2 or 4. In one embodiment, z is 2. In one embodiment, in conjugates III-b-1-1 and III-b-0-1, z is 2 or 4. In one embodiment, in conjugates III-b-1-1 and III-b-0-1, z is 4.

[0201] In one embodiment, B of the compound of formula (I) is the terminal group R 10 and the cleavable sequence 1 of L 1 binds to the payload to form a compound of formula (II), in which case B is absent in the molecule resulting from the binding of the cleavable sequence 1 and the payload. In such a case, M is LKa-L2―L1―B―P, where it can be understood that B is absent. In such a case, M can also be represented as LKa-L 2 ―L 1 ―P. In one embodiment, n is 3, L 2 is -(CH2) p -(CH2)2(CO)-, p is 3, L 1 is GGFG, B is -NH-CH2-U- or absent or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, U is O, and g is 1.

[0202] In one embodiment, conjugate III-a-0-1 has the structure: TIFF2025524725000118.tif58165, and wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n .

[0203] In one embodiment, conjugate III-a-0-2 has the structure: TIFF2025524725000119.tif53165, and wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n .

[0204] In one embodiment, conjugate III-a-0-3 has the structure: TIFF2025524725000120.tif63165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n .

[0205] In one embodiment, conjugate III-a-0-4 has the structure: TIFF2025524725000?121.tif99165, and wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n .

[0206] In one embodiment, conjugate III-a-0-5 has the structure: TIFF2025??524725000122.tif83165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n .

[0207] It should be noted that there seems to be a "?" in "TIFF2025??524725000122.tif83165" in the original text. If this is an error, it should be corrected for a more accurate translation.In one embodiment, conjugate III-a-1-1 has the structure: TIFF2025524725000123.tif63165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as follows.

[0208] In one embodiment, conjugate III-a-1-2 has the structure: TIFF2025524725000124.tif187165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as follows.

[0209] In one embodiment, conjugate III-a-1-3 has the structure: TIFF2025524725000125.tif114165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as follows.

[0210] In one embodiment, conjugate III-a-1-4 has the structure: TIFF2025524725000126.tif99165, wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as follows.

[0211] In one embodiment, i is 4, g is 1, and R 11 is methyl.

[0212] In one embodiment, n is 3, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, and L 1is GGFG, B is -NH-CH2-U-, and U is O. In one embodiment, conjugate III-b-1-1 has the structure: TIFF2025524725000127.tif73165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n In one embodiment, conjugate III-a-0-2 is as follows (conjugate III-a-0-2-1),

[0213] In one embodiment, conjugate III-a-0-2 is as follows (conjugate III-a-0-2-1), TIFF2025524725000128.tif52165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly)n.

[0214] In one embodiment, i is 4, j is 8, g is 1, and R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-1), TIFF2025524725000129.tif67165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n In one embodiment, i is 4, j is 12, g is 1, and R

[0215] is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-1), 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-1), TIFF2025524725000130.tif67165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n [[ID=:30]]In one embodiment, i is 4, j is 12, and R

[0216] In one embodiment, i is 4, j is 12, and R 11is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-2), TIFF2025524725000131.tif60165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as described.

[0217] In one embodiment, i is 4, j is 12, and R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-3), TIFF2025524725000132.tif61165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as described.

[0218] In one embodiment, n is 3, i is 4, j is 12, m is 1, and R 11 is methyl. In one embodiment, conjugate III-b-1-1 is as follows (conjugate III-b-1-1-4), TIFF2025524725000133.tif70165 wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n as described.

[0219] Preparation of Conjugates The conjugates of the present disclosure can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by ligase-catalyzed site-specific conjugation of a compound of formula (I) having a targeting molecule and a payload, and the targeting molecule is modified by a ligase recognition sequence. The method includes step A and step B.

[0220] Step A. Preparation of the Linking Unit-Payload Intermediate In a preferred embodiment, B of the compound of formula (I) is covalently bound to a payload containing another reactive group via a reactive group.

[0221] The linker - payload intermediate prepared using the compound of formula (I) of the present disclosure has a defined structure, defined composition, and high purity. Therefore, when a conjugation reaction with an antibody is carried out, the introduced impurities are reduced or no other impurities are introduced. When such an intermediate is used for ligase - catalyzed site - specific conjugation with a modified antibody containing a ligase recognition sequence, a homogeneous ADC with highly controllable quality is obtained.

[0222] Step B. Binding a targeting molecule to the compound of formula (I) having a payload The targeting molecule of the present disclosure can be conjugated to a compound of formula (I) having a payload (i.e., a compound of formula (II)) by any method known in the art.

[0223] The targeting molecule and the compound of formula (I) having a payload can be bound to each other via the ligase - specific recognition sequence of the substrate. The recognition sequence depends on the specific ligase used. In one embodiment, the targeting molecule is an antibody having a terminal modification based on a recognition sequence introduced at the C - terminus of the light chain and / or heavy chain, and the targeting molecule is conjugated to the compound of formula (II) under the catalysis of a wild - type or optimized engineered ligase or any combination thereof, and under suitable catalytic reaction conditions.

[0224] In a specific embodiment, the ligase is sortase A, and the conjugation reaction can be represented by the following scheme: TIFF2025524725000134.tif22165

[0225] The triangle represents the part of the antibody, and the pentagon represents the part of the compound of formula (II). n, X, and J are as defined above. G is the corresponding recognition sequence of the receptor substraten When conjugating, the peptide bond upstream of glycine in the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate binds to the free N-terminus of G n and a new peptide bond is formed. The resulting amino acid sequence is LPXTG n . The sequences G n and LPXTGJ are as defined above.

[0226] Metabolism of the conjugate in the physiological environment When some or all of the linker is cleaved within the tumor cell, the anti-tumor compound moiety is released, and the anti-tumor effect of the anti-tumor compound is exhibited. When the linker is cleaved at the drug-binding position, the anti-tumor compound is released in its native structure and exhibits its native anti-tumor effect.

[0227] In one embodiment, the cleavable sequence 1 (e.g., GGFG) can be cleaved by lysosomal enzymes (e.g., cathepsin B and / or cathepsin L).

[0228] In one embodiment, Sp1 includes a self-destructive spacer. In one embodiment, Sp1 includes PABC, acetal or heteroacetal. In one embodiment, L 1 is GGFG. In one embodiment, the linker includes -GGFG-NH-CH2-O-. In one embodiment, -GGFG-NH-CH2-O- represents a combination of a restriction enzyme site and a self-destructive spacer, which is cleaved intracellularly to release the molecule of interest (e.g., a drug).

[0229] Pharmaceutical compositions and pharmaceutical preparations Another object of the present disclosure is to provide a pharmaceutical composition comprising a prophylactically effective amount or a therapeutically effective amount of the conjugate of the present disclosure and at least one pharmaceutically acceptable carrier.

[0230] Another object of the present disclosure is to provide a pharmaceutical composition comprising a prophylactically effective amount or a therapeutically effective amount of the anti-TROP2 antibody of the present disclosure and at least one pharmaceutically acceptable carrier.

[0231] The pharmaceutical composition of the present disclosure can be administered in any manner as long as it achieves the effect of preventing, alleviating, precluding, or curing human or animal symptoms. For example, various suitable dosage forms, particularly, injection preparations, such as freeze-dried injection powders, injection solutions, or sterile injection powders, can be prepared according to the administration route.

[0232] The term "pharmaceutically acceptable" means that when in contact with the tissues of a patient within the scope of ordinary medical judgment, it does not cause excessive toxicity, irritation, allergic reaction, etc., has a reasonable benefit-risk ratio, and is effective for the intended purpose.

[0233] The term pharmaceutically acceptable carrier refers to a carrier material that does not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered physiological saline. Also, pharmaceutically acceptable carriers include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffers, toxicity adjusters, etc., and also include sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In some embodiments, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the active ingredient for treatment. Such pharmaceutical carriers can be sterile liquids, such as water and oils, such as petroleum, animal, vegetable, or synthetic origin oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous or glycerol solutions of glucose can also be used particularly as liquid carriers for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of wetting agent, emulsifier, or pH buffer, such as acetic acid, citric acid, or phosphoric acid.

[0234] In one embodiment, the pharmaceutical composition of the present disclosure has a drug-to-antibody ratio (DAR) that is an integer or non-integer from about 1 to about 20, such as from about 1 to about 10, from about 1 to about 8, from about 1 to about 6, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2.5, or from about 1 to about 2. In certain embodiments, the conjugate of the present disclosure has a DAR of about 2, about 4, about 6, or about 8.

[0235] Methods of Treatment and Use The conjugate or antibody is useful for treating tumors and / or autoimmune diseases. Tumors sensitive to conjugate treatment include those characterized by certain tumor-associated antigens or cell surface receptors, which can be recognized by the targeting molecule of the conjugate and killed by the payload / cytotoxin of the conjugate.

[0236] Accordingly, in yet another aspect, there is also provided the use of the conjugate or antibody of the present disclosure, or the pharmaceutical composition of the present disclosure, in the manufacture of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0237] In another aspect, there is provided the conjugate of the present disclosure or the pharmaceutical composition of the present disclosure for use in treating a tumor or an autoimmune disease.

[0238] In another aspect, there is provided the antibody of the present disclosure or the pharmaceutical composition of the present disclosure for use in treating a tumor or an autoimmune disease.

[0239] In a further aspect, there is provided a method of treating a tumor or an autoimmune disease, comprising administering to a subject in need thereof an effective amount of the conjugate or antibody of the present disclosure or the pharmaceutical composition of the present disclosure.

[0240] In a preferred embodiment, the conjugate of the present disclosure formed by the conjugation of an anti-human TROP2 antibody and a small molecule cytotoxin can specifically bind to TROP2 on the surface of tumor cells and can selectively kill TROP2-expressing tumor cells. In another preferred embodiment, there is provided the use of the conjugate (or antibody) of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, disorder or condition selected from TROP2-positive tumors. In a more preferred embodiment, the disease, disorder or condition is a TROP2-positive tumor. In one embodiment, the TROP2-positive tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, and the like.

[0241] The dosage of the conjugate (or antibody) administered to a subject can be adjusted to a considerable extent. The dosage may vary depending on the specific route of administration and the needs of the subject and may be subject to the judgment of a medical professional.

[0242] Beneficial effects The antibody-drug conjugate of the present invention uses a specially designed linker-payload, is more stable, can achieve high efficacy with a lower DAR, and thus can reduce side effects and increase the therapeutic index.

[0243] The present disclosure utilizes a binding unit having a unique structure and uses a ligase to catalyze the conjugation of a targeting molecule and a payload. The conjugate of the present disclosure has good uniformity, high activity, and high selectivity. Furthermore, since the toxicity of the binding unit-payload intermediate is much lower than that of the free payload, the drug manufacturing process is less harmful and advantageous for industrial production.

[0244] The conjugate of the present disclosure achieves at least one of the following technical effects: (1) High inhibitory activity against target cells, or a strong killing effect on target cells. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action). (4) Good safety (low toxicity to non-target normal cells or tissues, and / or fewer side effects, wider treatment range), etc. (5) High-level module design, easy assembly of multiple drugs.

Examples

[0245] Preparation Examples To more clearly explain the objectives and technical solutions, the present disclosure will be further described below with reference to specific examples. It should be understood that the examples are not intended to limit the scope of the present disclosure. Specific experimental methods not mentioned in the following examples were carried out according to conventional experimental methods.

[0246] Instruments, Materials and Reagents Unless otherwise specified, the instruments and reagents used in the examples are commercially available. The reagents can be used directly without further purification. MS: Thermo Fisher Q Exactive Plus, Water2795 - Quattro Micro Triple Quadrupole Mass Spectrometer HPLC: Waters 2695, Agilent 1100, Agilent 1200 Semi-preparative HPLC: Lisure HP plus 50D Flow Cytometry: CytoFLEX S HIC-HPLC: Butyl-HIC, Mobile Phase A: 25 mM PB, 2 M (NH4)2SO4 (pH 7.0), Mobile Phase B: 25 mM PB (pH 7.0), Flow Rate: 0.8 ml / min, Acquisition Time: 25 min, Injection Volume: 20 μg, Column Temperature: 25 °C, Detection Wavelength: 280 nm, Sample Chamber Temperature: 8 °C. SEC-HPLC: Column: TSK-Gel G3000 SWXL, TOSOH 7.8 mm ID × 300 mm, 5 μm, Mobile phase: 0.2 M KH2PO4, 0.25 M KCl (pH 6.2), Flow rate: 0.5 ml / min, Acquisition time: 30 min, Injection volume: 50 μl, Column temperature: 25 °C, Detection wavelength: 280 nm, Sample tray temperature: 8 °C. CHO was obtained from Thermo Fisher Scientific. pcDNA3.3 was obtained from Life Technology. HEK293F was obtained from Prejin. PEIMAX transfection reagent was obtained from Polyscience. MabSelect Sure ProA was obtained from GE. Capto S ImpAct was obtained from GE. Rink-amide-MBHA-resin and dichloro resin were obtained from Nankai synthesis. HCC1954 was obtained from ATCC CAT# CRL-2338. SK-BR-3 was obtained from ATCC CAT# HTB-30. BT-474 was obtained from ATCC CAT# HTB-20. NCI-N87 cells were obtained from ATCC CAT# CRL-5822. MCF7 was obtained from ATCC CAT#HTB-22; MDA-MB-231 was obtained from ATCC CAT#HTB-26. MDA-MB-468 was obtained from ATCC CAT#HTB-132. CFPAC-1 was obtained from ATCC CAT# CRL-1918. NCI-H2110 was obtained from ATCC CAT# CRL-5924. JIMT-1 was obtained from Wuxi Apptech. Capan-1 was obtained from ATCC CAT#CRL-1573. Optimized recombinant enzyme sortase A derived from Staphylococcus aureus was prepared in E. coli.

[0247] Example 1 Construction, Expression, Purification and Identification of Antibody Expression Vector 1.1 Construction of Expression Vector Encoding Anti-TROP2 Antibody To generate an expression vector encoding the light chain of the anti-TROP2 antibody, the nucleic acid sequence of LC was individually cloned into the pCDNA3.3 vector (Life Technology). To generate an expression vector encoding the heavy chain of the anti-TROP2 antibody, the nucleic acid sequence of HC was individually cloned into the pCDNA3.3 vector (Life Technology).

[0248]

Table 3

[0249] 1.2 Expression of anti-TROP2 antibody Plasmids encoding the light and heavy chains of the anti-TROP2 antibody were paired and mixed at a mass ratio of 2:1. The plasmid pair and PEIMAX (Polyscience) transfection reagent were separately diluted in HEK293F basal medium and then evenly mixed. The mixture was left at room temperature and added to the HEK293F seed cell culture. The cells were cultured at 32 for 24 hours, sampled for analysis of cell density and viability, and supplemented with 10% volume of HEK293F feed medium. Then, the culture temperature was changed to 32 °C for the next culture. At 72 hours from the start of incubation, the cell culture was sampled again for analysis of cell density and viability. At 144 hours from the start of incubation, the cell culture was sampled for analysis of cell density and viability.

[0250] 1.3 Purification of anti-TROP2 antibody The antibody was purified by affinity chromatography according to the manufacturer's instructions. Briefly, a chromatography column (BestChrom, Shanghai, China) was packed with MabSelect SureLX resin (GE Healthcare) and equilibrated with 50 mM Tris, 150 mM NaCl (pH 7.4). Then, the supernatant of the cell culture was obtained and applied to the column. The column was washed with 50 mM Tris, 150 mM NaCl (pH 7.4) to remove non-specifically bound proteins. Next, the antibody was eluted with 50 mM citrate buffer (pH 3.5), and the antibody-containing eluate was adjusted to pH 6.5 using 1 M Tris-HCl (pH 9.0). Finally, the buffer of the antibody was exchanged to 50 mM Tris, 150 mM NaCl (pH 7.4) using an Anicon Ultra-15 centrifugal filter (Merk Millipore).

[0251] 1.4 Binding reaction rate and affinity analysis The binding reaction rate and affinity analysis were performed. Surface plasmon resonance (SPR) analysis was carried out using a Biacore T200 (GE healthcare) with Sensor Chip Protein A (GE Healthcare) according to the manufacturer's instructions. All measurements were performed at 25 °C in HBS-EP + buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). Approximately 110 - 140 RU (resonance units) of each of the purified antibodies were captured on flow cells 2 and 4 (i.e., the reaction surfaces) of the sensor chip, respectively. Flow cells 1 and 3 were treated with HBS-EP + buffer to function as reference surfaces.

[0252] Serial dilutions (243, 81, 27, 9, 3, 1, 0.333, and 0.111 nM, respectively) of the recombinant extracellular domain of human TROP2 (i.e., the analyte) (Acrobio system) were prepared in HBS-EP +It was prepared with a buffer. After antibody capture, a serial dilution of TROP2 was injected for 3 minutes at a flow rate of 30 μL / min (binding step), and then buffer was flowed for 10 minutes (dissociation step). The chip surface was regenerated by performing two pulses of injecting 10 mM glycine-HCl (pH 1.5) at a flow rate of 50 μL / min for 30 seconds each. The collected data was processed using Biacore T200 Evaluation software by methods well-known in the art. The processing included the following steps: (1) setting the response to zero on the Y-axis and the start of injection to zero on the X-axis, (2) performing a two-step reference by first subtracting the reference surface data from the reaction surface data to obtain the analyte injection curve, and then subtracting the buffer injection curve from the analyte injection curve, and (3) performing kinetic analysis using a 1:1 binding model by global fitting. The results for each antibody were shown as Ka (on-rate), Kd (off-rate), and K D (equilibrium dissociation constant).

[0253] 1.5 SEC-HPLC detection of anti-TROP2 antibodies The antibody samples were centrifuged at 12,000 rpm for 5 minutes, and the supernatants were applied to an SEC-HPLC column to detect the proportions of monomer (corresponding to intact antibody), high molecular weight (HMW, corresponding to antibody aggregates due to aggregation), and low molecular weight (LMW, corresponding to antibody fragments due to degradation) forms of each antibody. TIFF2025524725000136.tif63165

[0254] Trop2 is widely expressed in normal tissues, and the affinities of Ab13 and Ab16 are decreased. Low-affinity antibodies should be able to improve safety while maintaining the efficacy verified in previous experiments.

[0255] 1.6 Internalization activity MDA-MB-468 with good viability was trypsinized, collected, suspended in cold FACS buffer (DPBS + 2% FBS), and 2×10 6Adjusted to cells / ml. Anti-Trop2 antibody and isotype control antibody samples were fluorescently labeled by mixing with anti-human IgG-Fc-AF647 secondary antibody at a molar ratio of 1:1 for 20 minutes at room temperature. The labeled antibody was added to the cell suspension at a final concentration of 10 μg / ml. The antibody-cell mixture was incubated on ice for 1 hour. After surface binding, the antibody-cell mixture was washed twice with cold FACS buffer to remove excess antibody. The cells were incubated at 37 °C for 0 minutes, 10 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, and 240 minutes for antibody internalization, and 1 × 10 5 cells per well were transferred to a V-bottom 96-well plate. Internalization was stopped by returning the cells to an ice bath. Non-internalized cells (0 minutes) were divided into MAX and MIN groups, and antibody-internalized cells were marked as group I. Groups MIN and I were washed twice with quenching buffer (150 mM NaCl + 100 mM glycine, pH = 2.0 - 2.5) to dissociate surface binding of the antibody. After quenching, all groups were washed twice with FACS buffer and analyzed on the APC channel by flow cytometry.

[0256] The MFI data was incorporated into the following formula, and the results were analyzed by a one-phase exponential binding function in Prism6. Isotype control: MFI (I) = MFI (試料、I) - MFI (アイソタイプ対照抗体、I) MFI (MAX) = MFI (試料、MAX) - MFI (陰性抗体、MAX) MFI (MIN) = MFI (試料、MIN) - MFI (陰性抗体、MIN) Internalization rate: R (t) =(MFI (I) - MFI (MIN) ) / MFI (MAX) × 100%

[0257] As shown in Figure 1, all four antibodies showed equivalent internalization activity against MDA-MB-468.

[0258] Example 2 Preparation of Intermediates Example 2.1 Preparation of Linker-Payload 1 and Linker-Payload 2 TIFF2025524725000137.tif50165opSu is a mixture with TIFF2025524725000138.tif32165

[0259] Preparation of Intermediate MC-GGFG-DXd Intermediate MC-GGFG-DXd is either commercially available or prepared according to the procedure described in EP2907824. Using this compound, Linker-Payload 1 is prepared.

[0260] Preparation of Linker-Payload Intermediate 1 Linker-Payload Intermediate 1 can be synthesized by conventional solid-phase polypeptide synthesis using Rink-amide-MBHA-resin. The amino acids of the binding unit were protected using Fmoc. The coupling reagent was selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the product was cleaved from the resin using a TFA / TIS / H2O solution. The product was purified by preparative HPLC, lyophilized and stored for use. MS m / z: [M-H] - = 1382.6.

[0261] Preparation of Linker-Payload 1 Linker-Payload Intermediate 1 and MC-GGFG-DXd (molar ratio approximately 1:2) were weighed, dissolved in water and DMF respectively, then thoroughly mixed to obtain a mixture, which was reacted at 0 - 40 °C for 0.5 - 30 hours. When the reaction was complete, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued at 0 - 40 °C for an additional 0.2 - 20 hours. After the reaction was complete, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain Linker-Payload 1. MS m / z: [(M + 3H) / 3] + = 1163.3.

[0262] Preparation of Linker-Payload 2 The following Linker-Payload Linker-Payload 2 can be prepared using the same synthetic routes and reagents as Linker-Payload 1. TIFF2025524725000140.tif74165opSu is a mixture with TIFF2025524725000141.tif32165.

[0263] Example 2.2 Preparation of Linker-Payload 3 and Linker-Payload 4 Preparation of Intermediate 11 TIFF2025524725000142.tif78165

[0264] Step A: N-(2-Bromo-5-fluorophenyl)acetamide: Concentrated H2SO4 (3 mL) was added to a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL), followed by the addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) portionwise at room temperature. The mixture was stirred for 3 hours and then poured into 2000 mL of ice water. A precipitate formed, which was collected by filtration and dried in vacuo at room temperature to give N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.68 (dd, J = 8.9, 6.0 Hz, 1H), 7.61 (ddd, J = 10.7, 5.3, 3.1 Hz, 1H), 7.02 (ddd, J = 8.9, 8.0, 3.1 Hz, 1H), 2.11 (s, 3H). MS m / z 232.0(M+H).

[0265] Step B: N-(5-Fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL) was added n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) dropwise over 1 h at -78 °C. After completion, the mixture was stirred under N2 for 0.5 h. Then, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise over 0.5 h at -78 °C, and the mixture was stirred from -78 °C to room temperature for 6 h. The mixture was poured into 500 mL of saturated aqueous NH4Cl at 0 °C, extracted with ethyl acetate (500 mL × 3), washed with brine (250 mL × 2), dried over Na2SO4, and concentrated. The mixture was triturated with (PE / EA = 1:1, 100 mL) for 10 min, filtered, and the cake was collected and dried in vacuo to give N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. LCMS m / z 206.1 (M - 18 + H), 246.1 (M + Na).

[0266] Step C: N-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) in CH2Cl2 (170 mL) and water (170 mL) were added silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol), and the mixture was stirred at 30 °C for 6 h. The mixture was filtered through celite, washed with CH2Cl2 (100 mL), the filtrate was concentrated, and purified by FCC (EA / PE = 0 - 40%) to give N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g) as a pale yellow solid. MS m / z 222.1 (M + H).

[0267] Step D: N-(3-Fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: 1-Butyl nitrite (8.48 g, 63.28 mmol) was added to a stirred mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) at 0 °C, followed by the addition of t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0 °C for 2 h. After completion, the mixture was acidified with HCl (2N) and adjusted to pH = 3. The mixture was extracted with ethyl acetate (200 mL × 3), washed with brine (100 mL × 2), dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 min, filtered, the cake was collected and dried in vacuo to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12 g) as a yellow solid. MS m / z 251.1 (M+H).

[0268] Step E: N,N'-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: 10% Pd / C (1 g) was added to a solution of acetic anhydride (90 mL) and THF (90 mL) of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (12 g, 47.96 mmol), and the mixture was stirred at 25 °C under a H2 atmosphere for 16 h. After cooling to 0 °C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0 °C for 1 h. It was filtered through celite, and the filtrate was poured into ice water (500 mL). It was extracted with ethyl acetate (500 mL × 3), washed with brine (250 mL × 2), dried over Na2SO4, and concentrated. The residue was triturated with tert-butyl methyl ether (120 mL) for 10 min, filtered, the cake was collected, and dried in vacuo to obtain N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g) as a yellow solid. MS m / z 279.1 (M+H).

[0269] Step F: N,N'-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: Aqueous HCl solution (2 N, 150 mL) was added to a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (7.9 g, 28.39 mmol) in MeOH (150 mL), and the mixture was stirred at 50 °C for 7 h. After cooling to 0 °C, saturated aqueous NaHCO3 solution was added dropwise to adjust the pH to 8, extracted with ethyl acetate (200 mL × 3), washed with brine (200 mL × 2), dried over Na2SO4, and concentrated under reduced pressure to obtain N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (6.0 g) as a yellow solid. 11H NMR (400 MHz, chloroform-d) δ 6.57 (s, 3H), 6.18 (td, J = 11.1, 2.4 Hz, 2H), 4.52 (dt, J = 13.3, 5.0 Hz, 1H), 3.13 (ddd, J = 17.5, 13.0, 4.6 Hz, 1H), 3.00 - 2.81 (m, 1H), 2.69 (dtd, J = 9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J = 13.0, 4.3 Hz, 1H). MS m / z 237.1 (M+H).

[0270] Step G: N-(8-Amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (4.0 g, 16.93 mmol) in DMF (80 mL) was added NCS (2.26 g, 16.93 mmol) portionwise at 0 °C. The mixture was stirred at room temperature for 16 h. The mixture was poured into 200 mL of ice water. A precipitate formed, which was collected by filtration and dried in vacuo at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 8.0 Hz, 1H), 7.71 (s, 2H), 6.62 (d, J = 11.9 Hz, 1H), 4.53 (ddd, J = 13.0, 8.0, 4.7 Hz, 1H), 3.18 - 3.04 (m, 1H), 2.91 (ddd, J = 17.5, 12.4, 4.8 Hz, 1H), 2.21 - 2.08 (m, 1H), 1.99 - 1.83 (m, 4H). MS m / z 271.0(M+H).

[0271] Step H: N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide: To a mixture containing N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g, 14.78 mmol) in toluene (400 mL) was added (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol), and o-cresol (10 mL). The mixture was heated to reflux under N2 for 24 h. The solvent was removed under reduced pressure and the mixture was purified by FCC (THF / CH2Cl2 = 0 - 60%) to give N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4.1 g) as a brown solid. MS m / z 498.1 (M+H).

[0272] Step I: A mixture containing (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione:N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of aqueous concentrated HCl was stirred at 70 °C for 36 h under N2. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid. MS (ESI) m / z 456.1 (M+H).

[0273] Preparation of Intermediate 12 (12-1, 12-2) TIFF2025524725000143.tif3716512-1 and 12-2 were prepared as TFA salts from (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (Intermediate 11) by preparative HPLC. TIFF2025524725000144.tif156165

[0274] Preparation of Linker-Payload 3 TIFF2025524725000145.tif187165opSu is TIFF2025524725000146.tif32165a mixture with

[0275] Preparation of Compound 13 (Step A) 4.33 g of Fmoc-Gly-Gly-OH and 6.84 g of Pb(OAc)4 were weighed and added to a 500 mL one-neck round-bottom flask. Anhydrous THF / toluene (120 / 40 mL) was added under a nitrogen atmosphere and stirred to dissolve. Then, 1.16 mL of pyridine was added to the reaction system. The reaction system was heated to 80 °C and refluxed for 5 hours under a nitrogen atmosphere. Samples were taken and detected by HPLC to monitor the reaction.

[0276] The reaction system was cooled to room temperature, filtered, and the filter cake was washed three times with EA. The filtrates were combined and concentrated to dryness. Column chromatography (PE:EA = 100:0~50:100) was carried out to obtain about 2000 mg of the target product as a white solid in a yield of 44%.

[0277] Preparation of Compound 15 (Step B) 200 mg of Compound 13 was weighed and added to a 100 mL one-neck round-bottom flask. Then, 15 mL of THF was added and stirred to dissolve. Then, Compound 14 (312 mg, 3.0 equivalents) and TsOH·H2O (15 mg, 0.15 equivalents) were added to the reaction system. The reaction system was reacted overnight at room temperature. Samples were taken and detected by TLC (PE / EA = 1:1) to monitor the reaction. The starting materials had generally disappeared and new points were detected.

[0278] A saturated sodium bicarbonate solution was added to quench the reaction. Extraction was carried out three times with EA. The organic phases were combined, washed with physiological saline, dried over anhydrous magnesium sulfate, and concentrated. The crude product was purified by column chromatography (PE:EA = 5:1~1:1) to obtain about 80 mg of the target product as a colorless oil in a yield of 29%. MS m / z: [M+H] + = 501.1

[0279] Preparation of Compound 16 (Step C) 200 mg of Compound 15 was weighed and added to a 100 ml one-neck round-bottom flask. Then, 10 ml of EtOH and 5 ml of EA were added and completely dissolved. Next, 40 mg of palladium carbon was added to the reaction system under a nitrogen atmosphere, and the reaction system was purged with hydrogen gas three times. The reaction system was kept under a hydrogen atmosphere and stirred at room temperature for 0.5 h. Samples were taken and the reaction was monitored by TLC (DCM / MeOH = 10:1). The raw material substance had almost disappeared and a new point was detected.

[0280] The reaction system was filtered and the filter cake was washed three times with EA. The filtrates were combined and concentrated to dryness to obtain 200 mg of the product as a white solid in 100% yield. The product could be used directly in the next reaction without purification. MS m / z: [M-H] - = 409.4.

[0281] Preparation of Compound 21 (Step D) Step D-1 2.0 g of dichlororesin was weighed and placed in a polypeptide synthesis tube. DCM (10 ml) was added and it was swollen at room temperature for 30 min. The solvent was removed by vacuum suction. The resin was washed twice with 7 mL of DCM (each washing time was 1 min). The solvent was removed by vacuum suction. Then, 200 mg of Compound 16 was weighed and added to a 50 ml centrifuge tube. DCM (about 10 ml) was added and the solid was dissolved by shaking and added to the above resin. Stirring was carried out to immerse all the resin in the solution (if the resin adhered to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 4 - 5 h. After the reaction was completed, an appropriate amount of methanol was added. Stirring was carried out for 30 min. The solvent was removed by vacuum suction. The resin was washed in turn with 10 mL of DMF once, methanol once, DMF once, methanol once, and DMF twice (each washing time was 1 min). The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was suitable for the next coupling step.

[0282] Step D-2 Deprotection was carried out twice by adding 10 mL of a ready-made 20% piperidine / DMF solution each time and reacting for 10 minutes. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed twice with 10 mL of DMF each, once with methanol, once with DMF, once with methanol, and twice with DMF in that order (each washing time was 1 minute). The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. Both the resin and the solution were dark blue.

[0283] 563 mg of Fmoc-Phe-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Then, about 7 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added and activated for 10 - 30 minutes to obtain an activated reaction solution.

[0284] 3 molar equivalents of the activated reaction solution were added to the resin. Stirring was carried out to completely immerse the resin in the solution (if the resin adhered to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the solvent was removed by vacuum suction. The resin was washed twice with 10 mL of DMF each, once with methanol, once with DMF, once with methanol, and twice with DMF in that order (each washing time was 1 minute). The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was suitable for the next coupling step.

[0285] Step D-3 The two deprotections were carried out by adding 10 mL of a ready-made 20% piperidine / DMF solution each time and reacting for 10 minutes. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed twice with 10 mL of DMF each time, once with methanol, once with DMF, once with methanol, and twice with DMF in sequence (each washing time was 1 minute). The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. Both the resin and the solution were dark blue.

[0286] 531 mg of Fmoc-GG-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Then, about 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added and activated for 10 - 30 minutes to obtain an activated reaction solution.

[0287] 3 molar equivalents of the activated reaction solution were added to the resin. Stirring was carried out to completely immerse the resin in the solution (if the resin adhered to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin was washed twice with 10 mL of DMF each time, once with methanol, once with DMF, once with methanol, and twice with DMF in sequence (each washing time was 1 minute). The solvent was removed by vacuum suction. A small amount of dry resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was suitable for the next coupling step.

[0288] Step D-4 The two deprotections were carried out by adding 10 mL of a ready-made 20% piperidine / DMF solution each time and reacting for 10 minutes. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed successively with 10 mL of DMF twice, methanol once, DMF once, methanol once, and DMF twice (each washing time was 1 minute). The solvent was removed by vacuum suction. A small amount of the dried resin was collected for ninhydrin detection. Both the resin and the solution were dark blue. Next, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube, and about 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIEA was added to the resin. Stirring was carried out to completely immerse the resin in the solution (if the resin adhered to the tube wall, a small amount of DCM was used to wash the tube wall). Stirring was carried out for 2 - 3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin was washed successively with 10 mL of DMF twice, methanol once, DMF once, methanol once, and DMF twice (each washing time was 1 minute). The solvent was removed by vacuum suction. A small amount of the dried resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating that it was suitable for the next coupling step.

[0289] Step D-5 The resin was washed twice with 10 mL of methanol. Next, the solvent was completely removed by vacuum suction. The resin was poured out and weighed. A dissolution buffer was prepared in a 250 mL conical flask (the ratio of TFE / DCM was 80% / 20%, and the volume was 7 - 8 times the weight of the peptide resin). The dissolution buffer was added to the peptide resin and shaken well. The resin was fully immersed in the dissolution buffer, and dissolution was carried out at room temperature for 2 - 3 hours. Next, the dissolution buffer was filtered using a simple filter made with a syringe, and the resin was washed with 1 - 2 mL of DCM and discarded. Then, 150 mL of pre-cooled anhydrous ether was added to the dissolution buffer, shaken well, and then left to stand for 20 - 30 minutes. Using a 50 mL centrifuge tube, the above system was centrifuged at 3500 rpm for 3 minutes in a centrifuge, and the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under ultrasonic waves, centrifuged at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was placed in a centrifuge tube and air-dried overnight, and then preparative purification was carried out to obtain 125 mg of the product as a white solid in a 40% yield. MS m / z: [M-H] - = 641.5.

[0290] Preparation of Compound 22 (Step E) 150 mg of starting material Compound 21 and 55 mg of TSTU were weighed and added to a 10 mL one-neck round-bottom flask. Anhydrous DMF (3 mL) was added under a nitrogen atmosphere and stirred for 20 minutes. Then, 18 mg of Compound 12-1 and 20 μL of DIEA were sequentially added to the reaction system. Stirring was carried out at room temperature for 2 - 8 hours under a nitrogen atmosphere. Samples were taken and detected by HPLC to monitor the reaction. The peak of the starting material completely disappeared, and a new peak was detected.

[0291] Preparative purification was carried out on the reaction system, the target product was collected, freeze-dried, and about 22 mg of the product was obtained as a yellowish solid. MS m / z: [M+H] + = 1081.0.

[0292] Preparation of Linker-Payload 3 (Step F) Compound 22 (30 mg) was weighed and added to a 10 mL single-neck round-bottom flask, and purified water (2 mL) was added. Stirring was carried out for dissolution. A DMF solution (2 mL) containing linker-payload intermediate 1 (19.5 mg) was added to the reaction system and stirred. After reacting overnight, the reaction was monitored using HPLC until all of the starting materials were converted to the intermediate. An appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was further carried out at 0 - 40 °C for 0.2 - 20 hours. The reaction was monitored by HPLC until all of the intermediates were consumed, and then quenched with an acetic acid solution.

[0293] Fractional purification was performed on the reaction system, the target product was collected, freeze-dried, and about 25 mg of linker-payload 3 was obtained as a yellowish solid. MS m / z: [(M + 3H) / 3] + = 1194.4.

[0294] Preparation of Linker-Payload 4 The following linker-payload 4 can be prepared using the same synthetic route and reagents as linker-payload 3. The structure of linker-payload 4 is as follows: TIFF2025524725000147.tif83165opSu is a mixture with TIFF2025524725000148.tif32165.

[0295] Example 2.3 Preparation of Linker-Payload 5 TIFF2025524725000149.tif140165 Step 1: Preparation of Intermediate Compound b Step 1.1 Preparation of NH2-Asp(OtBu)-Rink Amide Resin 400 g of Rink amide resin was weighed and thoroughly swollen with 2400 mL of DCM. 2400 mL of deprotection reagent was added to completely remove Fmoc, and then washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0296] 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, and the reaction mixture was stirred with nitrogen at room temperature for 2 - 5 h and then filtered. The resin was successively washed with DMF and DCM and showed colorless or light yellow in the subsequent ninhydrin test.

[0297] 2400 mL of the deprotection reagent was added to completely remove Fmoc, and then it was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed blue.

[0298] Step 1.2 Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, and the reaction mixture was stirred with nitrogen at room temperature for 2 - 4 h and then filtered. The resin was successively washed with DMF and DCM and showed colorless or light yellow in the subsequent ninhydrin test.

[0299] 2400 mL of the deprotection reagent was added to completely remove Fmoc, and then it was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed blue.

[0300] Step 1.3 Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin, and the reaction mixture was stirred with nitrogen at room temperature for 2 - 4 h and then filtered. The resin was successively washed with DMF and DCM and showed colorless or light yellow in the subsequent ninhydrin test.

[0301] 2400 mL of the deprotection reagent was added to completely remove Fmoc, and then it was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0302] Step 1.4 Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 hour, it was slowly added to the reaction kettle together with the resin, and the reaction mixture was stirred with nitrogen at room temperature for 2 - 4 hours and then filtered. The resin was continuously washed with DMF and DCM and showed a colorless or light yellow color in the subsequent ninhydrin test.

[0303] 2400 mL of the deprotection reagent was added to completely remove Fmoc, and then it was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0304] Step 1.5 Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 hour, it was slowly added to the reaction kettle together with the resin, and the reaction mixture was stirred with nitrogen at room temperature for 2 - 4 hours and then filtered. The resin was continuously washed with DMF and DCM and showed a colorless or light yellow color in the subsequent ninhydrin test.

[0305] Step 1.6 Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 2400 mL of de-Dde reagent was added, and the reactants were stirred at room temperature under nitrogen for 0.5 h, then filtered. This process was repeated three times. After that, the resin was washed successively with DMF and DCM and showed blue in the subsequent ninhydrin test.

[0306] Step 1.7 Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT were weighed and dissolved in 2000 mL of DMF and 80.0 mL of DIC solution. After placing it in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle together with the resin. The reactants were stirred with nitrogen at room temperature for 2 - 4 h and then filtered. The resin was washed successively with DMF and DCM and showed colorless or light yellow in the subsequent ninhydrin test. The resin peptide was washed three times with absolute ethanol, filtered, and waited for cleavage.

[0307] Step 1.8 Preparation of intermediate compound b 10000 mL of cleavage reagent (TFA:TIS:H₂O = 95:2.5:2.5) was added to a 10 L reactor and cooled to -10 ± 2 °C. The dried and weighed resin was added. The reactants were warmed to room temperature and stirred under nitrogen for 2 - 3 h. Then, the resin was filtered and washed once with 100 mL of TFA. The filtrate and the washing solution were combined.

[0308] 40 L of pre-cooled (-less than 10 °C) cold ether was added to the product solution. The mixture was stirred for 10 min, and then the precipitate was centrifuged. After centrifugation, the supernatant was discarded, the precipitate was collected, washed with cold ether, and the precipitate was centrifuged again (each time, the centrifugation speed was set at 3600 rpm, the centrifugation time was 5 min, and the temperature of the centrifugation cavity was -5 °C).

[0309] The precipitate was collected as crude compound b. The crude product was purified by preparative HPLC and lyophilized to obtain pure compound b.

[0310] Step 2: Preparation of Intermediate Compound a TIFF2025524725000150.tif89165 Step 2.1 Preparation of Compound 2 Compound 1 (1 equivalent) and DMF (5 v / v) were added to a reaction flask, and the mixture was stirred and dissolved under nitrogen protection. After cooling the ice bath to 0 - 5 °C, DIEA (3 equivalents) was added dropwise. After the dropwise addition, the mixture was stirred at 5 °C for 10 minutes. Then, benzyl bromide (1.3 equivalents) was added dropwise. After the completion of the dropwise addition, it was allowed to warm naturally to room temperature of about 20 °C and stirred for 16 hours.

[0311] The reaction solution was slowly poured into ice water, MTBE was added and stirred, and the solution was allowed to stand for separation. The aqueous phase was extracted 4 times with MTBE. The combined organic phases were washed with saturated brine, then the organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain a yellow crude oil, which was applied to a column by the wet method. Elution with PE / EA = 6:1 gave a pale yellow oil (yield was 100%).

[0312] Step 2.2 Preparation of Compound 4 Under nitrogen protection, intermediate 2 (2.0 equivalents), compound 3 (1 equivalent), and THF (10 v / v) were added to a reaction flask and stirred to dissolve. TsOH (0.1 equivalent) was weighed and added to the reaction. The reaction was maintained at 20 - 22 °C for 4 hours. The reaction solution was slowly poured into ice water, extracted 3 times with EA. The combined organic phases were successively washed with saturated aqueous sodium bicarbonate solution, water, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain a crude product. The product was collected by mixing the silica gel sample through a column with elution of PE / EA = 1:1, concentrated, and a white solid was obtained with a yield of 40%.

[0313] Step 2.3 Preparation of Compound 7 Under nitrogen protection, Compound 4 and DMAc (10 v / v) were added to a reaction flask and stirred to dissolve. The reaction mixture was cooled to 14 - 18 °C, and DBU (0.5 equiv) was added dropwise. The reaction mixture was stirred at this temperature for 1.5 h, and the completion of the reaction of the starting materials was monitored by TLC. The reaction mixture was cooled to 0 - 5 °C, and PPTS (0.5 equiv), EDCI (1 equiv), HOBT (1 equiv), and Compound 6 (0.85 equiv) were added. The reaction was carried out at 0 - 10 °C for 3 - 4 h, and the reaction was monitored by LCMS.

[0314] The reaction solution was added to ice water, 2 - methyltetrahydrofuran was added for extraction once, and the aqueous phase was extracted twice with 2 - methyltetrahydrofuran. The combined organic phases were washed with 0.5 M hydrochloric acid, saturated aqueous NaHCO3, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column chromatography. The product was collected by elution with DCM / MeOH and concentrated under vacuum to obtain a white solid in 78% yield.

[0315] Step 2.4 Preparation of Compound 10 Under nitrogen protection, Intermediate 7 and DMAc (10 v / v) were added to a reaction flask and stirred to dissolve. The reaction mixture was cooled to 14 - 18 °C, and DBU (0.5 equiv) was added dropwise. The reaction mixture was stirred at this temperature for 1.5 h, and the completion of the reaction was monitored by TLC. The reaction mixture was cooled to 0 - 5 °C, and PPTS (0.5 equiv), EDCI (1 equiv), HOBT (1 equiv), and Compound 9 (0.85 equiv) were added. The reaction was carried out at 0 - 10 °C for 3 - 4 h, and the reaction was monitored by LCMS.

[0316] The reaction solution was added to ice water, 2 - methyltetrahydrofuran was added for extraction once, and the aqueous phase was extracted twice with 2 - methyltetrahydrofuran. The combined organic phases were washed with 0.5 M hydrochloric acid, saturated aqueous NaHCO3, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column chromatography. The product was collected by elution with DCM / MeOH and concentrated under vacuum to obtain a white solid in 50% yield.

[0317] Step 2.5 Preparation of Compound a Under nitrogen protection, Intermediate 10 was dissolved in DCM (15 v / v), and DBU (0.5 equivalent) was added dropwise at 20 °C. The reaction mixture was stirred at 18 - 22 °C for 5 hours. The completion of the reaction was monitored by LCMS. The reaction solution was diluted with DCM and purified by column using the wet method, and the product was collected by elution with DCM:MeOH to obtain a white solid in 82% yield.

[0318] Step 3: Preparation of Intermediate Compound c TIFF2025524725000151.tif84165 Compound b (400 mg, 0.245 mmol) and Compound a (377 mg, 0.539 mmol) were dissolved in DMF (6 ml). Then, DIPEA (159 mg, 1.23 mmol) and HATU (233 mg, 0.613 mmol) were added to the reaction solution, and the reaction mixture was stirred at room temperature for 2 hours. After Compound b disappeared, it was purified by preparative HPLC, and the preparation solution was lyophilized to obtain 380 mg of the product in 52% yield. C 142 H 207 O 49 N 21 [(M + 3H) / 3] + Calculated value for: 997.8, Measured value: 875.9 (fragmented mass).

[0319] Step 4: Preparation of Intermediate Compound d TIFF2025524725000152.tif89165 Compound c (380 mg, 0.245 mmol) was dissolved in purified water (80 ml), and palladium hydroxide (38 mg) was added. The system was exchanged with hydrogen three times, and the reaction mixture was stirred at room temperature for 1.5 hours. During this period, the progress of the reaction was monitored, and the reaction was stopped immediately after the disappearance of the starting material to prevent the increase of the de-Fmoc product. The reaction solution was filtered and purified by preparative HPLC to obtain 270 mg of the product in 76% yield. C 128 H 195 O 49 N 21 [(M + 3H) / 3] +Calculated value for: 937.8, measured value: 875.9 (fragmented mass).

[0320] Step 5: Preparation of intermediate compound e TIFF2025524725000153.tif94165 Compound d (270 mg, 0.096 mmol) and 12-1 (120 mg, 0.211 mmol) were dissolved in DMF (5 ml), then DIPEA (62 mg, 0.48 mmol) and HATU (92 mg, 0.24 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for 2 - 16 hours. After completion of the reaction monitored by HPLC, the reaction mixture was directly purified by preparative HPLC, the collected eluents were combined, and lyophilized to obtain 235 mg of the product in 66% yield. C 174 H 229 O 55 Cl2F2N 27 [(M + 3H) / 3] + Calculated value for: 1229.2, measured value: 1229.3.

[0321] Step 6: Preparation of linker-payload 5 TIFF2025524725000154.tif99165 Compound e (210 mg, 0.057 mmol) was dissolved in DMF (5 ml), then diethylamine (0.5 ml) was added, and the reaction was allowed to proceed at room temperature for 15 minutes, with the reaction endpoint monitored by HPLC. After completion of the reaction, it was adjusted to neutral with 10% aqueous TFA under an ice bath, and the reaction was purified by preparative HPLC and lyophilized to obtain 145 mg of the product in 73% yield. C 159 H 219 O 53 Cl2F2N 27 [(M + 3H) / 3] + Calculated value for: 1155.2, measured value: 1155.3.

[0322] Example 3 Preparation of Targeted Molecule-Pharmaceutical Conjugate 3.1 Preparation of DS1062a Analog and Trodelvy DS1062a Analog (DS1062a and DS-1062 *) was prepared based on the method described in Patent US20160297890A or was produced by WuXi Biologics.

[0323] Trodelvy was commercially available.

[0324] 3.2 Preparation of ADC-1 3.2.1 Treatment of GQhRS7 GQhRS7 was treated with ultrafiltration, dialysis, or a desalting column. The storage solution was exchanged with the ligase buffer.

[0325] 3.2.2 Enzymatic Catalytic Coupling of ADC-1 ADC-1 was prepared by the coupling reaction of GQhRS7 and linker-payload 1 under the catalysis of wild-type sortase A or a mutant ligase optimized and operated based on it. In the ligase buffer, the modified antibody and linker-payload were completely mixed at a molar ratio of 1:1 to 1:100 and added to the solid-phase coupling system. The solid-phase coupling system contained a ligase immobilized on the matrix of the solid-phase coupling system. The immobilized ligase catalyzed the coupling reaction of antibody GQhRS7 with linker-payload 1. The coupling reaction was carried out at 4 - 40 °C for 0.5 - 20 hours. After the reaction was completed, ultrafiltration or dialysis was performed on the reaction mixture to remove the unreacted intermediates and obtain ADC-1. ADC-1 was stored at 4 °C or -80 °C in a buffer containing 20 mM citric acid and 200 mM NaCl (pH 5.0).

[0326] 3.2.3 Detection and Analysis of ADC-1 by HIC-HPLC The DAR (drug-to-antibody ratio) distribution of ADC-1 was analyzed by HIC-HPLC. The antibody GQhRS7 without cytotoxic was less than 5%. The coupled product mainly contained ADC-1 and the DAR was 3.5.

[0327] 3.2.4 Detection and Analysis of ADC-1 by SEC-HPLC The degree of high-molecular-weight aggregation of ADC-1 was analyzed by SEC-HPLC. The results showed that no high-molecular-weight polymer was detected in ADC-1. This indicated that the coupling reaction conditions were mild and did not cause damage to the antibody structure.

[0328] 3.3 Each linker-payload intermediate was site-specifically conjugated to an antibody by ligase to form an ADC. The method for the conjugation reaction can be found in WO2015165413A1. The resulting ADCs are as listed in the following table: TIFF2025524725000155.tif64165

[0329] 3.4 Binding activity Human Trop2 ECD at a concentration of 0.5 μg / mL was coated onto a 96-well plate at 4 °C overnight. Next, the plate was blocked with 3% BSA-PBST at room temperature for 1 hour. After washing with PBST (0.05% Tween), a series of samples of test articles containing different concentrations of ADC-2 (i.e., ADC2), Trodelvy, and GQhRS7 were each added to the 96-well plate and then incubated at room temperature for 60 minutes. After incubation, goat anti-human FC secondary antibody (HRP) (Sinobiological, SSA001) was added at a ratio of 1:100000 and incubated again at room temperature for 60 minutes. After washing, the plate was treated with TMB solution (Sigma, T0440) as the HRP substrate and the reaction was stopped with 1 M H2SO4. The absorbance of each well was detected at a wavelength of 450 nm.

[0330] The results of this analysis are shown in Figure 1.2. These results demonstrate that ADC-2 has a binding affinity similar to that of GQhRS7 and Trodelvy.

[0331] 3.5 Internalization activity NCI-N87 with good survival rate was trypsinized, collected, and suspended in cold FACS buffer (DPBS + 2% FBS). The cells were incubated on ice for 1 hour with the test drug solution at a final concentration of 50 μg / ml. The antibody-cell mixture was washed twice with cold FACS buffer to remove excess antibody. The cells bound with antibody were fluorescently labeled by mixing with the ice-cold anti-human-IgG-Fc-AF647 secondary antibody solution diluted 500-fold for 30 minutes. After fluorescent labeling, the antibody-cell mixture was washed twice again. For test drug internalization, the cells were incubated at 37°C for 10 minutes, 30 minutes, 60 minutes, 90 minutes, 150 minutes, and 210 minutes. The cells were suspended with quenching buffer (150 mM NaCl + 100 mM glycine (pH = 2.0 - 2.5)) to dissociate the antibody bound to the cell surface. After acidification, the cells were washed twice with cold FACS buffer and analyzed by flow cytometry in the APC channel.

[0332] The MFI data was incorporated into the following formula, and the results were analyzed by a one-phase exponential association function in Prism8. Internalization amount: A = MFI(I) - MFI(MIN). Internalization rate: R = [MFI(I) - MFI(MIN)] / [MFI(MAX) - MFI(blank)] × 100%

[0333] As shown in Figure 1.3, ADC2 shows internalization activity equivalent to DS1062, Trodelvy, and GQhRS7 in NCI-N87.

[0334] Effect Example 1 Bystander killing effect of conjugate in BxPC-3 / HepG2 Effect Example 1.1 Bystander killing effect of ADC-1 on HepG2 in BxPC-3 / HepG2 co-culture assay The cell concentrations of Trop2-positive cells BxPC-3 and Trop2-negative cells HepG2 were 1x10 6Adjusted to cells / mL, 200 μL per well (cell volume BxPC-3:HepG2 = 4:1) was seeded into a 6-well plate and supplemented with 2.8 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium. The cells were incubated overnight at 37 °C and 5% CO2 in a cell incubator. 3 mL of 20 nM of ADC-1 and DS1062a were added to the cells cultured overnight respectively (the final drug concentration was 10 nM per well). A negative control group was set up: 3 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium was added to each well. After treatment, the cells were transferred to the incubator and incubated for 96 hours. After incubation, the cells were digested, washed once with 1X PBS, then transferred to a flow tube and centrifuged at 2000 rpm for 3 minutes. Next, the supernatant was discarded and the cell amount and cell viability were detected. A specific amount of cells was washed with 1X PBS, the supernatant was discarded after centrifugation, 200 μL of 100 nM anti-human Trop2 antibody was added, the cells were mixed, and incubated at 4 °C for 30 minutes. The cells were washed with 1X PBS, the supernatant was discarded after centrifugation, 200 μL of 5 μg / mL human IgG Fc antibody was added, and the obtained cells were continuously incubated at 4 °C for 30 minutes after mixing. Finally, the cells were washed with 1X PBS, the supernatant was discarded after centrifugation, the cells were resuspended in PBS, detected by flow cytometry, and analyzed by FlowJo software. The results are shown in Table 4.

[0335]

Table 4

[0336] Effect Example 1.2 Bystander Killing Effect of ADC-2 on HepG2 in the BxPC-3 / HepG2 Co-Culture Assay The cell concentrations of Trop2-positive cells BxPC-3 and Trop2-negative cells HepG2 were 1x10 6Adjusted to cells / mL, 300 μL per well (cell volume BxPC-3:HepG2 = 2:1) was seeded into a 6-well plate and supplemented with 2.7 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium. The cells were incubated overnight at 37 °C and 5% CO2 in a cell incubator. 3 mL of 20 nM of ADC-2 and DS1062a were added to the cells cultured overnight respectively (the final drug concentration was 10 nM per well). Furthermore, in the following process, the same process as above was used to evaluate the bystander killing effect of ADC-2 and DS1062a. The results are shown in Table 5.

[0337] [Table 5]

[0338] Effect Example 1.3 Bystander killing effect of ADC-6 on HepG2 in the BxPC-3 / HepG2 co-culture assay Using the same process as in Effect Example 1.2, the bystander killing effects of the control (dato-pomab), DS1062a, ADC-1, ADC-2, ADC-4, and ADC-6 were evaluated. The results are shown in Table 6.

[0339] [Table ......

[0340] Conclusion The experimental results of Effect Example 1.1 showed that both ADC-1 and the DS1062a analog had a bystander killing effect, and there was no significant difference in their effectiveness.

[0341] The experimental results of Effect Example 1.2 showed that the bystander killing effect of ADC-2 was better than that of DS1062a.

[0342] The experimental results of Effect Example 1.3 showed that all of ADC-1, ADC-2, ADC-4, ADC-6 and DS1062a analogs had a bystander killing effect. The bystander killing effects of ADC-2 and ADC-6 were better than that of DS1062a. The bystander killing effect of ADC-6 was significantly better than that of ADC-4.

[0343] Effect Example 2: Effect of conjugates targeting TROP2 on cell proliferation Effect Example 2.1: Inhibitory effects of ADC-7 and ADC-1 on tumor cell proliferation Effect Example 2.1.1: Inhibitory effect of ADC-7 on the proliferation of human pharyngeal squamous cell carcinoma FaDu 2000 human pharyngeal squamous cell carcinomas FaDu with high TROP2 expression were seeded in a 96-well plate containing 100 μL of medium per well and incubated overnight in a cell incubator at 37 °C and 5% CO2. 100 μL of different concentrations (200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, 0.000512 nM, and 0.0001024 nM) of ADC-7 and DS1062a were added to each well (3 replicate wells for each concentration). A positive control group was set up: 100 μL of puromycin was added to each well at a concentration of 10 μg / mL. A negative control group was set up: 100 μL of the complete medium of FaDu cells was added to each well. After the administration was completed, the cells were transferred to the incubator and incubated for 96 hours. The 96-well plate was taken out from the cell incubator at 37 °C and equilibrated to room temperature for 30 minutes. After discarding the medium, 100 μL of DMEM and 50 μL of CellTiter Glo reagent were added to each well, and the cells were shaken at 200 rpm in the dark for 15 minutes, and the luminescence signal reflecting the cell viability was detected by a microplate reader. The inhibitory effects of the test drugs on the proliferation of human pharyngeal squamous cell carcinoma FaDu are shown in Figure 2 and Table 7.

[0344]

Table 7

[0345] Effect Example 2.1.2 Inhibitory Effect of ADC-1 on the Proliferation of Human Pancreatic Cancer Cells BxPC-3 The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human pancreatic cancer cells BxPC-3 (100 μL per well containing 2000 cells), and the inhibitory effect of ADC-1 was evaluated using the same process. The inhibitory effect of the test drug on the proliferation of human pancreatic cancer cells BxPC-3 is shown in Figure 3 and Table 8.

[0346] [Table 8]

[0347] Effect Example 2.1.3 Inhibitory Effect of ADC-1 on the Proliferation of Human Breast Cancer Cells MDA-MB-468 The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human breast cancer cells MDA-MB-468 (100 μL per well containing 4000 cells), and the inhibitory effect of ADC-1 was evaluated using the same process. The inhibitory effect of the test drug on the proliferation of human breast cancer cells MDA-MB-468 is shown in Figure 4 and Table 9.

[0348] [Table 9]

[0349] Effect Example 2.1.4 Inhibitory Effect of ADC-7 on the Proliferation of Human Gastric Cancer Cells NCI-N87 The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human gastric cancer cells NCI-N87 (100 μL per well containing 5000 cells), and the inhibitory effect of ADC-7 was evaluated using the same process. The inhibitory effect of the test drug on the proliferation of human gastric cancer cells NCI-N87 is shown in Figure 5 and Table 10. [Table 10]

[0350] Effect Example 2.2 Inhibitory Effects of ADC-2 and ADC-3 on Tumor Cell Proliferation Effect Example 2.2.1 Inhibitory Effects of ADC-2 and ADC-3 on the Proliferation of Human Pharyngeal Squamous Cell Carcinoma FaDu The test drugs ADC-1 and DS1062a in Example 2.1.1 were replaced with ADC-2, ADC-3, and ADC-1, and the inhibitory effects were evaluated using the same process. The inhibitory effects of the test drugs on the proliferation of human pharyngeal squamous cell carcinoma FaDu are shown in Figure 6 and Table 11.

[0351] [Table 11]

[0352] Effect Example 2.2.2 Inhibitory Effects of ADC-2 and ADC-3 on the Proliferation of Human Pancreatic Cancer Cell BxPC-3 The human pharyngeal squamous cell carcinoma FaDu in Example 2.2.1 was replaced with human pancreatic cancer cell BxPC-3, and the inhibitory effect of ADC-2 was evaluated using the same process. The inhibitory effects of the test drugs on the proliferation of human pancreatic cancer cell BxPC-3 are shown in Figure 7.1 and Table 12.

[0353] [Table 12]

[0354] Effect Example 2.3 Inhibitory Effects of ADC-2 and DS-1062a on the Proliferation of BxPC-3, FaDu, and NCI-N87 Cytotoxicity assays were performed using Trop2-positive cancer cells BxPC-3 (Figure 7.2), FaDu (Figure 7.3), and NCI-N87 (Figure 7.4) to analyze the effect of the conjugate on tumor cell proliferation. The test drugs included conjugates ADC2, DS1062a, and GQhRS7. Briefly, 3000 - 5000 cells were seeded in 96-well plates and allowed to adhere overnight. The cells were treated with the designated drugs at various concentrations for 168 hours. Cell viability was examined by the CellTiter-Glo® Luminescent Cell Viability Assay, and the percentage of cell viability was calculated.

[0355] In Trop2-positive BxPC-3, FaDu, and NCI-N87, ADC2 showed more potent cytotoxicity than DS1062a. The IC 50 value of ADC2 was lower than that of DS1062a (see the following table). TIFF2025524725000165.tif46165

[0356] Conclusion The results of Effect Example 2.1.1 showed that both ADC-7 and DS1062a were able to inhibit the proliferation of FaDu cells, and the inhibitory effect of ADC-7 was slightly better.

[0357] The results of Effect Example 2.1.2 showed that both ADC-1 and DS1062a were able to inhibit the proliferation of BxPC-3 cells.

[0358] The results of Effect Example 2.1.3 showed that both ADC-1 and DS1062a were able to inhibit the proliferation of MDA-MB-468 cells.

[0359] The results of Effect Example 2.1.4 showed that both ADC-7 and DS1062a were able to inhibit the proliferation of NCI-N87 cells.

[0360] The results of Effect Example 2.2.1 showed that ADC-2, ADC-3, and ADC-1 could inhibit the growth of FaDu cells.

[0361] The results of Effect Example 2.2.2 showed that ADC-2, ADC-3, and ADC-1 could inhibit the growth of BxPC-3 cells, and the inhibitory effect of ADC-2 was slightly better than those of ADC-3 and ADC-1.

[0362] The results of Effect Example 2.3 showed that ADC-2 could inhibit the growth of BxPC-3 cells, FaDu cells, and NCI-N87 cells, and the inhibitory effect of ADC-2 was better than that of DS1062a.

[0363] Effect Example 3 In vivo Efficacy Evaluation Test Effect Example 3.1 In vivo Efficacy Evaluation of ADC-1 Effect Example 3.1.1 In vivo Efficacy Evaluation of ADC-1 Against BxPC-3 Cells I. BxPC-3 cells in the logarithmic growth phase were collected, and the cell density was adjusted to 10x10 6 cells / mL with Matrigel buffer (PBS:Matrigel = 1:1). 0.2 mL of the prepared BxPC-3 cell suspension was subcutaneously injected into the right scapula of 6- to 8-week-old SPF female BALB / c nude mice.

[0364] II. The tumor diameter was measured with calipers, and the tumor volume was calculated according to the formula V = 0.5axb 2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor). Six days after cell inoculation, when the average tumor volume reached about 151 mm 3 , the animals were randomly divided into a vehicle control group, a 3 mg / kg group of DS1062a, and a 3 mg / kg group of ADC-1, with 6 animals in each group. The animals in each group were administered by tail vein injection, and the vehicle control group was administered an equal volume of vehicle. The tumor volume of the animals in each group was measured twice a week within 35 days after administration, and the tumor volume of the animals on the 35th day was compared among the groups. The T / C and TGI values were calculated using the tumor volume. The calculation formulas are as follows: T / C% = T RTV / C RTV ×100% (TRTV : RTV and C of the treatment group RTV : (RTV of the vehicle control group). The relative tumor volume (RTV) was calculated based on the results of tumor measurement, and the calculation formula is RTV = V t / V0, where V0 is the average tumor volume measured at the time of grouping (i.e., D0), and V t is the average tumor volume at one measurement, and T RTV and C RTV used the data of the same day. Calculation of TGI(%): TGI(%) = [1 - (average tumor volume at the end of administration of the treatment group - average tumor volume at the start of administration of the treatment group) / (average tumor volume at the end of treatment of the vehicle control group - average tumor volume at the start of treatment of the vehicle control group)] × 100%.

[0365] III. After 35 days of administration, the average tumor volumes of the 3 mg / kg group of ADC-1 (T / C = 49.05%, TGI = 57.56%, p = 0.002) and the 3 mg / kg group of DS1062a (T / C = 64.05%, TGI = 40.54%, p = 0.010) were 648 mm 3 and 847 mm 3 respectively. The results are shown in Figure 8 and Table 13.

[0366]

Table 13

[0367] Conclusion According to the results of Effect Example 3.1.1, both the 3 mg / kg group of ADC-1 and the 3 mg / kg group of DS1062a were able to significantly inhibit tumor growth compared with the control group.

[0368] Effect Example 3.1.2 In vivo efficacy evaluation of ADC-1 against NCI-N87 gastric cancer cells Replace the BxPC-3 cells in Example 3.1.1 with NCI-N87 gastric cancer cells. Seven days after cell inoculation, the average tumor volume was approximately 227 mm 3When it reached this point, the animals were randomly divided into a vehicle control group, a 3 mg / kg group of IMMU-132 (Trodelvy), a 3 mg / kg group of DS1062a, and a 3 mg / kg group of ADC-1, with 6 animals in each group. The in vivo efficacy evaluation of ADC-1 was evaluated using the same process as in Effect Example 3.1.1. The results are shown in Figure 9 and Table 14.

[0369]

Table 14

[0370] Conclusion After 32 days of administration, the 3 mg / kg group of ADC-1 (T / C = 57.17%, TGI = 55.09%, p < 0.001) and the 3 mg / kg group of DS1062a (T / C = 49.98%, TGI = 60.23%, p < 0.001) had average tumor volumes of 697 mm 3 and 658 mm 3 respectively, and both were able to significantly inhibit tumor growth.

[0371] Effect Example 3.1.3 In Vivo Efficacy Evaluation of ADC-1 Against Human Breast Cancer BR-05-0028 The human breast cancer BR-05-0028 model (IHC 3+) was obtained from tumor samples resected by clinical surgery. The tumor samples were inoculated into nude mice of the P0 generation, and the tumor tissue used in this example was of the P5 generation. Tumor tissue with a volume of about 30 mm 3 was subcutaneously inoculated into the right hind back of 6-8-week-old SPF female Balb / c nude mice. 28 days after tumor tissue inoculation, when the average tumor volume reached about 171 mm 3 the animals were randomly divided into a vehicle control group, a 5 mg / kg group of IMMU-132, a 5 mg / kg group of DS1062a, and a 5 mg / kg group of ADC-1, with 6 animals in each group. The in vivo efficacy evaluation of ADC-1 was evaluated using the same process as in Effect Example 3.1.1. The results are shown in Figure 10 and Table 15.

[0372] [[ID=3—1]]

Table 15

[0373] Conclusion After 21 days of administration, the 5 mg / kg groups of IMMU-132 (T / C = 0.35%, TGI = 111.76%, p = 0.037), DS1062a (T / C = 0.48%, TGI = 111.60%, p = 0.037), ADC-1 (5 mg / kg group, T / C = 0.23%, TGI = 111.88%, p = 0.037) and ADC-1 (10 mg / kg group, T / C = 0.23%, TGI = 111.88%, p = 0.037) were able to significantly inhibit tumor growth.

[0374] Effect Example 3.2 In vivo Efficacy Evaluation of ADC-2 and ADC-3 Effect Example 3.2.1 In vivo Efficacy Evaluation of ADC-2 and ADC-3 Against BxPC-3 Cells Six days after cell inoculation, when the average tumor volume reached approximately 151 mm 3 the animals were randomly divided into a vehicle control group, a 3 mg / kg group of DS1062a, a 3 mg / kg group of ADC-1, a 3 mg / kg group of ADC-2, and a 3 mg / kg group of ADC-3, with 6 animals in each group. The in vivo efficacy evaluation of ADC-2 and ADC-3 was evaluated using the same process as in Effect Example 3.1.1. The results are shown in Figure 11 and Table 16.

[0375] [Table 16]

[0376] Conclusion After 28 days of administration, the 3 mg / kg group of ADC-2 (T / C = 12.72%, TGI = 104.54%, p = 0.001) and the 3 mg / kg group of DS1062a (T / C = 58.49%, TGI = 48.52%, p = 0.049) had average tumor volumes of 116 mm 3 and 540 mm 3 respectively, indicating that the 3 mg / kg group of ADC-2 could significantly inhibit tumor growth.

[0377] Effect Example 3.2.2 In Vivo Efficacy Evaluation of ADC-2 and ADC-3 Against NCI-N87 Gastric Cancer Cells Replace the BxPC-3 pancreatic cancer cells in Example 3.2.1 with NCI-N87 gastric cancer cells. Eight days after cell inoculation, when the average tumor volume reached approximately 188 mm 3 animals were randomly divided into a vehicle control group, a 3 mg / kg group of DS1062a, a 3 mg / kg group of ADC-1, a 3 mg / kg group of ADC-2, and a 3 mg / kg group of ADC-3, with 6 animals in each group. The in vivo efficacy evaluation of ADC-2 was performed using the same process as in Effect Example 3.2.1. The results are shown in Figure 12 and Table 17.

[0378]

Table 17

[0379] Conclusion After 28 days of administration, the 3 mg / kg group of ADC-2 (T / C = 14.08%, TGI = 122.25%, p < 0.001), the 3 mg / kg group of ADC-3 (T / C = 33.43%, TGI = 94.72%, p < 0.001), the 3 mg / kg group of ADC-1 (T / C = 46.15%, TGI = 76.62%, p < 0.001), and the 3 mg / kg group of DS1062a (T / C = 40.30%, TGI = 84.96%, p < 0.001) had average tumor volumes of 89, 212, 293, and 255 mm 3 respectively, indicating that all the test drugs could significantly inhibit NCI-N87 tumor growth.

[0380] Effect Example 3.2.3 In Vivo Efficacy Evaluation of ADC-2 and ADC-3 Against Human Pharyngeal Squamous Cell Carcinoma FaDu Replace the BxPC-3 cells in Example 3.2.1 with human pharyngeal squamous cell carcinoma FaDu. Eleven days after cell inoculation, when the average tumor volume reached approximately 123 mm 3When this was achieved, the animals were randomly divided into a vehicle control group, a 3 mg / kg group of DS1062a, a 3 mg / kg group of ADC-1, a 3 mg / kg group of ADC-2, and a 3 mg / kg group of ADC-2, with 6 animals in each group. The in vivo efficacy evaluation of ADC-2 was evaluated using the same process as in Effect Example 3.2.1. The results are shown in Figure 13.1 and Table 18.

[0381]

Table 18

[0382] Conclusion After 28 days of administration, the 3 mg / kg group of ADC-2 (T / C = 0.00%, TGI = 107.90%, p = 0.003), the 3 mg / kg group of ADC-3 (T / C = 0.25%, TGI = 107.63%, p = 0.003), the 3 mg / kg group of ADC-1 (T / C = 1.23%, TGI = 106.59%, p = 0.003) and the 3 mg / kg group of DS1062a (T / C = 1.68%, TGI = 106.11%, p = 0.003) had average tumor volumes of 0, 4, 21 and 28 mm respectively. 3 This showed that all the test drugs could significantly inhibit FaDu tumor growth. Furthermore, at the end of the experiment, 6 mice in the 3 mg / kg group of ADC-2 and 3 mice in the 3 mg / kg group of ADC-3 showed complete tumor regression respectively.

[0383] Effect Example 3.2.4 In vivo Efficacy Evaluation of ADC-2 against MDA-MB-468 MDA-MB-468 tumor cells (ATCC, HTB-132) were maintained in vitro at 37°C in an atmosphere of 0% CO2 in air as monolayer cultures in L-15 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation. For the in vivo antitumor effect test, 10x10 in 0.2 mL of PBS (1:1) containing Matrigel 6Individual MDA-MB-468 human breast cancer cells (Trop2 positive) were subcutaneously inoculated into the right flank of BALB / c nude mice. 24 days later, when the tumor volume reached an average of 187 mm 3 3 , mice with tumors were assigned and administered ADC-2 at 0.5 mg / kg, 1.5 mg / kg, and 4.5 mg / kg, Trodelvy at 4.5 mg / kg, and DS1062a at 4.5 mg / kg intravenously. The tumor volume was measured with calipers twice a week. The T / C and TGI values were calculated using the tumor volume.

[0384] Conclusion After 35 days of administration, the 4.5 mg / kg group of ADC-2 (T / C = 0.00%, TGI = 137.93%), the 4.5 mg / kg group of DS1062a (T / C = 1.35%, TGI = 136.06%), and the 4.5 mg / kg group of Trodelvy (T / C = 78.73%). The results are shown in Figure 13.2 and the following table. ADC-2 showed significantly better efficacy than Trodelvy and slightly better efficacy than DS1062a. TIFF2025524725000172.tif67165

[0385] Effect Example 3.3 In vivo Efficacy Evaluation of ADC-5 and ADC-6 Effect Example 3.3.1 In vivo Efficacy Evaluation of ADC-5 and ADC-6 Against BxPC-3 Pancreatic Cancer Cells Six days after cell inoculation, when the average tumor volume reached approximately 159 mm 3 3 , the animals were randomly divided into a vehicle control group, a 3 mg / kg group of ADC-5, a 3 mg / kg group of ADC-6, and a 3 mg / kg group of DS1062a, with 6 animals in each group. The in vivo efficacy evaluation of ADC-5 and ADC-6 was evaluated using the same process as in Effect Example 3.1.1. The results are shown in Figure 14 and Table 19.

[0386]

Table 19

[0387] Conclusion After 42 days of administration, the 3 mg / kg group of ADC-5 (T / C = 0.75%, TGI = 116.20%, p = 0.003), the 3 mg / kg group of ADC-6 (T / C = 2.48%, TGI = 114.17%, p = 0.003), and the 3 mg / kg group of DS1062a (T / C = 27.69%, TGI = 84.60%, p = 0.007) had average tumor volumes of 8 mm 3 , 27 mm 3 , and 302 mm 3 , respectively, indicating that all the test drugs could significantly inhibit tumor growth. The inhibitory effects of ADC-5 and ADC-6 were much better than those of DS1062a.

[0388] Effect Example 3.3.2 In vivo Efficacy Evaluation of ADC-5 and ADC-6 against NCI-N87 Gastric Cancer Cells Replace the BxPC-3 pancreatic cancer cells in Example 3.3.1 with NCI-N87 gastric cancer cells. Six days after cell inoculation, when the average tumor volume reached approximately 196 mm 3 , the animals were randomly divided into a vehicle control group, a 3 mg / kg group of ADC-5, a 3 mg / kg group of ADC-6, and a 3 mg / kg group of DS1062a, with 6 animals in each group. The in vivo efficacy evaluation of ADC-5 and ADC-6 was evaluated using the same process as in Effect Example 3.3.1. The results are shown in Figure 15 and Table 20.

[0389]

Table 20

[0390] Conclusion After 42 days of administration, the 3 mg / kg group of ADC-5 (T / C = 10.88%, TGI = 106.06%, p < 0.001), the 3 mg / kg group of ADC-6 (T / C = 3.89%, TGI = 114.36%, p < 0.001), and the 3 mg / kg group of DS1062a (T / C = 35.67%, TGI = 76.58%, p < 0.001) had average tumor volumes of 133 mm 3 , 48 mm 3 , and 437 mm 3It was shown that all test drugs could significantly inhibit tumor growth. The inhibitory effects of ADC-5 and ADC-6 were much better than those of DS1062a.

[0391] Effect Example 3.3.3 In vivo Efficacy Evaluation of ADC-5 and ADC-6 against Human Pharyngeal Squamous Cell Carcinoma FaDu Replace the BxPC-3 pancreatic cancer cells in Example 3.3.1 with human pharyngeal squamous cell carcinoma FaDu. Ten days after cell inoculation, when the average tumor volume reached about 119 mm 3 at this time point, the animals were randomly divided into a vehicle control group, a 2 mg / kg group of ADC-5, a 2 mg / kg group of ADC-6, and a 2 mg / kg group of DS1062a, with 6 animals in each group. The in vivo efficacy evaluation of ADC-5 and ADC-6 was evaluated using the same process as in Effect Example 3.3.1. The results are shown in Figure 16 and Table 21.

[0392]

Table 21

[0393] Conclusion After 31 days of administration, the 2 mg / kg group of ADC-5 (T / C = 11.34%, TGI = 94.56%, p = 0.004), the 2 mg / kg group of ADC-6 (T / C = 0.05%, TGI = 106.60%, p = 0.004), and the 2 mg / kg group of DS1062a (T / C = 15.47%, TGI = 90.15%, p = 0.005) had average tumor volumes of 217 mm 3 , 1 mm 3 , and 296 mm 3 respectively, indicating that all test drugs could significantly inhibit tumor growth. The inhibitory effect of ADC-6 was much better than that of DS1062a.

[0394] Effect Example 4 Serum Stability of ADC-1 An appropriate amount of Trop2 was conjugated to CNBr-activated agarose microspheres by covalent coupling to form an immobilized antigen. After blocking, a stable sample pre-incubated with plasma was added at a certain ratio and incubated with shaking. ADC-1 and DS1062a (ADC drugs) in the matrix were specifically captured by the immobilized antigen to form a solid-phase antigen / antibody complex, and unbound substances were removed by washing. After incubation, N-glycosidase was used to excise the coupled sugar chains in the Fc region of the ADC drug antibody, and then the ADC was recovered by formic acid elution, and the DAR was detected by LC-MS. The results are shown in Figure 17.

[0395] Conclusion The results showed that the serum stability of ADC-1 was significantly better than that of DS1062a.

Claims

**Claim 1** Formula (III): (In the formula,[[]] Q is hydrogen, -C ２ H ４ -(PEG) ｔ -(CO)NH ２ or LKb-P, M is hydrogen or LKa - LKb―P, each LKa is independently selected from and opSu is or a mixture thereof, each B is independently absent or the following (1) and (2): Each LKb is, independently, L ２ - L １ - B and provided that Q and M are not hydrogen at the same time, (1) a self-destructive spacer Sp1, and (2) a bond, or -CR １ R ２ -, C １－１０ alkylene, C ４－１０ cycloalkylene, C ４－１０ one or a combination of two or more of the divalent groups selected from heterocyclylene and -(CO)- is a combination, and preferably, B is -NH-CH ２ -U- or does not exist, or -NH-CH ２ -U-(CR １ R ２ ) ｇ -(CO)-, U does not exist, or is O, S, or NH, preferably O or S, m is an arbitrary integer from 1 to 3, P is a payload that binds to the B portion or the L １ portion of formula (III), and Each L １ is a cleavable sequence 1 that independently contains an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 contains 1 to 10 amino acids, Each L ２ is independently a bond, or C ２－２０ alkylene, and one or more of the -CH ２ - structures in the alkylene are -CR ３ R ４ -, -O-, -(CO)-, -S(=O) ２ -, -NR ５ - C ４－１０ cycloalkylene, C ４－１０ heterocyclylene, phenylene, or may be replaced, said cycloalkylene, heterocyclylene, and phenylene, each independently, unsubstituted or halogen, -C １－１０ alkyl, -C １－１０ haloalkyl, -C １－１０ alkylene-NH-R ８ and -C １－１０ alkylene-O-R ９ is substituted with at least one substituent selected from, Ld2 and each Ld1 are independently a bond or -NH-C １－２０ alkylene-(CO)-, -NH-(PEG) ｉ -(CO)-, or are independently unsubstituted or have a side chain of -(PEG) ｊ -R １１ and are natural amino acids or natural oligomeric amino acids having a degree of polymerization of 2 to 10 and substituted with -(PEG) ｔ - and -(PEG) ｉ - and -(PEG) ｊ - each is a PEG fragment containing the indicated number of consecutive -(O-C ２ H ４ )- structural units or consecutive -(C ２ H ４ -O)- structural units, and has an optional additional C １－１０ alkylene at one end R １ 、 R ２ 、 R ３ 、 R ４ 、 R ５ 、 R ６ 、 R ７ 、 R ８ 、 R ９ are each independently hydrogen, halogen, -C １－１０ alkyl, -C １－１０ haloalkyl, C ４－１０ cycloalkylene, or R １ and R ２ together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl group, or R ３ and R ４ together with the carbon atoms to which they are attached form a 3- to 6-membered cycloalkyl group, R １１ is C １－１０ alkyl, and n is an arbitrary integer from 2 to 20, d is 0 or an arbitrary integer from 1 to 6, each i is independently an integer from 0 to 100, preferably from 0 to 20, preferably each i is independently an integer from 0 to 12, more preferably from 0 to 8, particularly 4, each j is independently an integer from 1 to 100, preferably from 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, each t is independently an integer from 1 to 100, preferably from 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, z is an integer from 1 to 20)) A is an anti-TROP2 antibody or an antigen-binding fragment thereof, preferably modified to bind to the G of formula (III), and G is glycine, ｎ and is bound to the moiety, A conjugate having the structure of **Claim 2** The conjugate according to claim 1, having the structure of the following formula (III-a) or formula (III-b): **Claim 3** Having the following structure: preferably, z is an integer from 1 to 4, preferably 2, each i, i1, i2, i3, i4 is independently an integer from 0 to 100, preferably from 0 to 20, preferably each i, i1, i2, i3, i4 is independently an integer from 0 to 12, more preferably from 0 to 8, particularly 4, each j is independently an integer from 1 to 100, preferably from 1 to 20, preferably each j is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, each t is independently an integer from 1 to 100, preferably from 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, m is an arbitrary integer from 1 to 3, particularly 1 or 2, The conjugate according to claim 1 or 2. Preferably, n is 3, and L ２ is -(CH ２ ) ｐ -(CH ２ ) ２ (CO)-, or -(C ２ H ４ -O) ｐ -(CH ２ ) ２ (CO)-, p is 2 to 4, L １ is Gly-Gly-Phe-Gly, B is -NH-CH ２ -U-, or does not exist, or -NH-CH ２ -U-(CR １ R ２ ) ｇ -(CO)-, U does not exist, or U is O, g is 1, **Claim 4** [[ID=2 The payload is a cytotoxin or a fragment thereof, and is optionally derivatized for attachment to the B moiety or the L moiety in the compound of formula (III) as defined in claim 1 １ and is optionally derivatized for attachment to the B moiety or the L moiety in the compound of formula (III) as defined in claim 1 Preferably, the cytotoxin is a taxane, a maytansinoid, an auristatin, an epothilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, an indole-sulfonamide, a vinca alkaloid, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin 10 and analogs, halichondrin B, eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laurylmaltide, camptothecin and its derivatives, mitoxantrone, mitoguazone, nitrogen mustard, nitrosoureas, aziridine, benzodopa, carboquone, meturedepa, uredepa, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthramycin, bleomycin, actinomycin C, calvisine, calminomycin, cardinophilin, calminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferric adriamycin, rhodrubicin, lufocromomycin, streptozocin, dinostatin, zorubicin, trichothecene, T-2 toxin, verracurin AA), selected from the group consisting of basiloporin A, anguinine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethyl folate, methotrexate, pteropterin, trimethoprim, edatrexate, fludarabine, 6-mercaptopurine, thiampurine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyuridine, doxifluridine, floxuridine, calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitor, and proteasome inhibitor, and / or Selected from vinblastine, colchicine, taxanes, auristatins, maytansinoids, calicheamicins, doxorubicin, duocarmycin, SN-38, cryptophycin analogs, delstecan, duocarmazine, calicheamicin, centanamycin, drastansine, pyrrolobenzodiazepines, exatecan, and derivatives thereof, and / or Selected from auristatins, particularly MMAE, MMAF or MMAD, and / or Selected from exatecan and its derivatives, such as DX8951f, The conjugate according to any one of claims 1 to 3.

5. Wherein the payload is of formula (i): (Wherein a ＊ is 0 or 1, and p1 ＊ and p2 ＊ each of the carbon atoms marked with ＊ and ＊ is an asymmetric center, and the asymmetric center is in the S configuration, R configuration, or racemate L １＊ is unsubstituted or substituted with one substituent selected from halogen, —OH, and —NH ２ selected from C １－６ alkylene, M ＊ is -CH ２ -, -NH- or -O-, and L ２＊ is an alkylene C １－３ and R １＊ and R ２＊ are each independently hydrogen, C １－６ alkyl, halogen, and C １－６ (selected from alkoxy) Having the structure of, the conjugate according to any one of claims 1 to 4.

6. L １＊ is selected from a linear alkylene, a branched alkylene, a cyclic alkylene, and a cyclic alkyl-C １－６ linear alkylene group, and each of the alkylene and the cyclic alkylene is independently unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH １－６ A branched alkylene, a cyclic alkylene, and a C ３－６ cyclic alkylene and C ３－４ cyclic alkyl-C １－２ linear alkylene group, and the alkylene and the cyclic alkylene are each independently unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH ２ Preferably, L １＊ is selected from C １－４ alkylene, and the alkylene is unsubstituted or substituted with one substituent selected from halogen, -OH, and -NH ２ More preferably, L １＊ is -CH ２ -, -C ２ H ４ -, selected from, each of which is independently unsubstituted or substituted with at least one substituent selected from halogen, -OH, and -NH ２ and most preferably, L is substituted with at least one substituent selected from １＊ -CH ２ - Selected from, wherein "#" indicates the position bonded to the carbonyl, the conjugate according to claim 5.

7. a ＊ The conjugate according to any one of claims 5 to 6, wherein a is 0.

8. R １＊ is selected from C １－６ alkyl and halogen, and preferably, R １＊ is methyl or Cl. The conjugate according to any one of claims 5 to 7.

9. R ２＊ is selected from C １－６ alkyl, halogen, and preferably, R ２＊ is F, the conjugate according to any one of claims 5 to 8.

10. Wherein the payload is Selected from In particular, Selected from, the conjugate according to any one of claims 1 to 3.

11. Selected from Each g is independently an integer from 1 to 6, preferably from 1 to 3, more preferably 1, Each R １ and R ２ is independently selected from hydrogen, halogen, -C １－１０ alkyl, -C １－１０ haloalkyl, C ４－１０ cycloalkylene, or R １ and R ２ together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkyl group, and preferably, R １ and R ２ are hydrogen, Each t is independently an integer from 1 to 100, preferably from 1 to 20, preferably each t is independently an integer from 1 to 12, more preferably from 8 to 12, particularly 8 or 12, m is an arbitrary integer from 1 to 3, particularly 1 or 2, z is an integer from 1 to 20, particularly 2 or 4, more preferably 2, The conjugate according to any one of claims 1 to 3.

12. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V Ｈ ) and a light chain variable region (V Ｌ ), and The said V Ｈ is (i) X １ X ２ GMX ３ (SEQ ID NO: 1) (where X １ is N, T or A, X ２ is Y or A, X ３ is N or Q) containing the amino acid sequence of HCDR1, (ii) WINTX ４ X ５ GX ６ PX ７ YX ８ X ９ DFKG (SEQ ID NO: 2) (where X ４ is Y, H or D, X ５ is T or S, X ６ is E or V, X ７ is T or K, X ８ is T or A, X ９ is D or E) and contains the amino acid sequence of HCDR2 (iii) X １０ GFGS SYWYFDV (SEQ ID NO: 3) (where X １０ is G or S) containing an HCDR3 amino acid sequence Comprising, and / or The foregoing V Ｌ is (i) LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVS TAVA (SEQ ID NO: 14), (ii) LCDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 15), and (iii) LCDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) Comprising The conjugate according to claim 1.

13. The aforesaid V Ｈ is (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 Comprising, and / or The aforesaid V Ｌ is (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 (including), or The aforesaid V Ｈ is (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12 (including), and / or The aforesaid V Ｌ is (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 (including), or The said V Ｈ is (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 (including), and / or The aforesaid V Ｌ is (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 (including), or The aforesaid V Ｈ is (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 (including), and / or The aforesaid V Ｌ is (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 (including), or The said V Ｈ is (i) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 (including), and / or The foregoing V Ｌ is (i) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 (including), The conjugate according to claim 12.

14. Said V Ｈ comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequences of SEQ ID NOs: 21-25, and / or Said V Ｌ comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, The conjugate according to claim 12 or 13.

15. The antibody or antigen-binding fragment is a heavy chain constant domain (CH) comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, and / or a light chain constant domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29 (including), the conjugate according to any one of claims 12 to 14.

16. The conjugate according to any one of claims 12 to 15, wherein the antibody or antigen-binding fragment binds to TROP2 with an equilibrium dissociation constant (K Ｄ ) of about 0.5 nM to about 20 nM.

17. The conjugate according to claim 12, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequences of SEQ ID NOs: 30 to 33, and / or a light chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO:

35.

18. The conjugate according to claim 17, wherein the antibody or the antigen-binding fragment thereof comprises a C-terminal modification of the heavy chain and / or a C-terminal modification of the light chain such that the antibody, Sp, and the recognition sequence of the ligase donor substrate are sequentially linked, Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, and the recognition sequence of the ligase donor substrate is LPXTGJ, where X can be any natural or non-natural single amino acid, and J is an amino acid fragment that is absent or contains 1 to 10 amino acids.

19. The modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NOs: 30 to 33 and / or a light chain of SEQ ID NO: 40 or SEQ ID NO: 41, or The modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NOs: 36 to 39 and / or a light chain of SEQ ID NO: 34 or SEQ ID NO: 35, The conjugate according to claim 18.

20. The conjugate according to claim 1, having an integer or non-integer drug-to-antibody ratio (DAR) of 1 to 19.

21. Heavy chain variable region (V Ｈ ) and a light chain variable region (V Ｌ ), an anti-TROP2 antibody or an antigen-binding fragment thereof, wherein The aforesaid V Ｈ is (i) X １ An amino acid sequence of AGMN (where X １ is N or A) containing HCDR1, (ii) an HCDR2 comprising the amino acid sequence of WINTDSGEPTYTDDFKG (SEQ ID NO: 10) or WINTYTGEPTYTDDFKG (SEQ ID NO: 8), (iii) an HCDR3 comprising the amino acid sequence of GGGFGSSYWYFDV (SEQ ID NO: 11) and / or The foregoing V Ｌ is (i) an LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13), (ii) an LCDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 15), (iii) an LCDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) comprising, the anti-TROP2 antibody or antigen-binding fragment thereof.

22. said V Ｈ comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and / or Said V Ｌ comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27, The antibody or antigen-binding fragment according to claim 21.

23. a heavy chain constant domain (CH) comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, and / or A light chain constant domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29 The antibody or antigen-binding fragment according to claim 21, comprising the same. **Claim 24** An antibody or antigen-binding fragment according to claim 21, which binds to TROP2 with an equilibrium dissociation constant (K Ｄ ) of from about 0.5 nM to about 20 nM. **Claim 25** A heavy chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 32, and / or a light chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35, The antibody or antigen-binding fragment according to claim 21, comprising the same. **Claim 26** A polynucleotide or vector comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of claims 21 to 25. **Claim 27** A host cell comprising the polynucleotide or vector according to claim 26. **Claim 28** A pharmaceutical composition comprising a prophylactically effective amount or a therapeutically effective amount of the conjugate according to any one of claims 1 to 20, the antibody or antigen-binding fragment according to any one of claims 21 to 25, the polynucleotide or vector according to claim 26, or the host cell according to claim 27, and at least one pharmaceutically acceptable carrier. **Claim 29** Use of the conjugate according to any one of claims 1 to 20, the antibody or antigen-binding fragment according to any one of claims 21 to 25, or the pharmaceutical composition according to claim 28 in the manufacture of a medicament for treating a disease, wherein the disease is a TROP2-related tumor. **Claim 30** The use according to claim 29, wherein the TROP2-related tumor comprises a tumor overexpressing TROP2 or a tumor having a TROP2 gene mutation. **Claim 31** The use according to claim 29, wherein the tumor comprises breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial cancer. **Claim 32** A method for treating a subject suffering from a disease or preventing disease progression, comprising administering the conjugate according to any one of claims 1 to 20, the antibody or antigen-binding fragment according to any one of claims 21 to 25, or the pharmaceutical composition according to claim 28, wherein the disease is a tumor. **Claim 33** An antibody-drug conjugate comprising the antibody according to any one of claims 21 to 25. **Claim 34** A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 21 to 25, comprising (i) culturing the host cell according to claim 27 under conditions suitable for the expression of the antibody or its antigen-binding fragment; (ii) recovering the antibody or its antigen-binding fragment from the host cell or its cell culture; The method comprising the above.

Images