The combination of antibody-drug conjugates and anti-PD-1 antibodies, and their use.

Abstract

This disclosure relates to an antibody-drug conjugate combined with an anti-PD-1 antibody, and to its use.

Description

[Technical Field]

[0001] This disclosure relates to the biopharmaceutical field, particularly to antibody-drug conjugates and combinations with anti-PD-1 antibodies, and their use. [Background technology]

[0002] background TROP2 is a transmembrane protein that 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 autoogenesis, proliferation, and transformation. Experiments have shown that TROP2 can promote tumor growth, and that knockout of the TROP2 gene inhibits tumor cell growth. Due to the limited tissue expression of TROP2, the toxicity of treatment is reduced, which is also an advantage of targeting TROP2 therapy. Several TROP2 variants targeting ADCs have been proposed (e.g., DS-1062, TRODELVY, BAT8003). These therapies have significantly improved survival rates in patients with TROP2-positive cancers.

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

[0004] However, there is still a high demand for novel ADC drugs targeting TROP2, and highly safe ADCs are also one of the development directions of novel drugs.

Summary of the Invention

[0005] overview In the first aspect, a compound of formula (I): TIFF2026515759000002.tif32165 is provided In the formula, W is hydrogen, LKb, or -C2H4-(PEG) , , , 4~10 , 2 , , 10 , 1 , 2 , 10 , , , 4~10 , , 1~10 , , 1 , , 1 , ,<000,0013>, 10 -(CO)NH2, Y is hydrogen or LKa-LKb, provided that W and Y are not hydrogen simultaneously, Each LKa is independently TIFF2026515759000003.tif27165 selected from, and opSu is TIFF2026515759000004.tif32165 or a mixture thereof, Each LKb is independently L 2 ―L 1 ―B, Each B is independently the terminal group R 10 or, the following: 1) a self-cleaving spacer Sp1, 2) a bond, or -CR 1 R 2 -, C 1~10 alkylene, C 4~10 cycloalkylene, C 4~10 heterocyclylene, and -(CO)-, one of the divalent groups selected from or a combination of two or more of the divalent groups, and 3) the terminal group R​​​​​​​​​​​​​​2~20 It is an alkylene, where one or more -CH2- structures in the alkylene are -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, TIFF2026515759000005.tif4128,C 4~10 Cycloalkylene, C 4~10 It may be replaced by heterocyclylene or phenylene, where cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or halogen, -C 1~10 Alkyl, -C 1~10 Haloalkyl, -C 1~10 Alkilen-NH-R 8 and -C 1~10 Alkilen-OR 9 It is substituted with at least one substituent selected from the following: Ld2 and each Ld1 are independently bonded or -NH-C 1~20 Alkylene-(CO)-,-NH-(PEG) i- (CO)- is selected from, or is a natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or natural oligomeric amino acid is independently unsubstituted or has -(PEG) in its side chain. j -R 11 It has been replaced with, -(PEG) t -,-(PEG) i - and - (PEG) j -Each contains 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 A PEG fragment containing alkylene R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 These are, independently, hydrogen, halogen, and -C.1~10 Alkyl, -C 1~10 Haloalkyl, C 4~10 Selected from cycloalkylenes, or R 1 and R 2 They, together with the carbon atoms to which they are bonded, form a 3-6 membered cycloalkyl group, or R 3 and R 4 Together with the carbon atoms to which they are bonded, they form a 3-6 membered cycloalkyl group. R 11 is C 1~10 It is alkyl, m is any integer between 1 and 3. n is any integer between 2 and 20. d is 0 or any integer from 1 to 6, and 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 between 1 and 100, preferably between 1 and 20, and preferably each j is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Each t is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each t is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12.

[0006] In the second embodiment, equation (II): A compound having the structure of TIFF2026515759000006.tif32165 is provided. During the ceremony, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P M is hydrogen or LKa-LKb-P. However, Q and M are not hydrogen at the same time. P is the B portion or L portion of the compound in formula (I). 1 It is a payload that is joined to a part, n, d, Ld1, Ld2, t, LKa, and LKb are defined as in equation (I), Preferably, M is hydrogen or LKa-L 2 -L 1 -B-P, where each B independently does not exist or is one of the following: 1) a self-destructive spacer Sp1, and 2) a combination of a bond or -CR 1 R 2 -, C 1~10 alkylene, C 4~10 cycloalkylene, C 4~10 heterocyclylene, and -(CO)-, or a combination of two or more of said divalent groups, and 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 even more preferably Sp1 is -O-CH2-U- or -NH-CH2-U-, where -O- or -NH- is bonded to the cleavable sequence 1 and U does not exist or is 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 X1AGMN (SEQ ID NO: 45), 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) 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 the fourth aspect, a cytotoxin having the structure of formula (i): TIFF2026515759000007.tif68165 is provided, wherein, a* is 0 or 1, The carbon atoms marked with p1* and p2* are each an asymmetric center, and the asymmetric center is in the S configuration, R configuration, or racemic form, L 1* is unsubstituted C 1~6 alkylene, or C 1~6 alkylene substituted with one substituent selected from halogen, -OH, and -NH2, 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 the fifth aspect, a combined pharmaceutical comprising a conjugate and an anti-PD-1 antibody is provided, and the conjugate has the structure of formula (III): TIFF2026515759000008.tif37165, 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 TIFF2026515759000009.tif27165, opSu is TIFF2026515759000010.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 non-existent or the following: 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)-, or a combination of two or more of said divalent groups, preferably B is -NH-CH2-U, non-existent, -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, or NH-CH2-U-(CH2) g -(CO)-, provided that Q and M are not hydrogen simultaneously, <00​​​​​​​​​​​​​​​​​​​​​​​​​​Heterocyclylene, which may be replaced by phenylene, 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 is substituted with at least one substituent selected from the group consisting of: -(PEG) t -, -(PEG) i - and -(PEG) j - each contains the indicated number of consecutive -(O-C2H4)-structural units or consecutive -(C2H4-O)-structural units and has an optional additional C 1~10 alkylene at one end and is a PEG fragment, 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, and 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, and 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 between 1 and 100, preferably between 1 and 20, preferably independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Each g is an integer between 1 and 10, for example, 1, 2, 3, 4, 5, 6, 7. A is the G of compound (I) n It is an anti-TROP2 antibody or its antigen-binding fragment that binds to a portion, where G is glycine. z is an integer between 1 and 20.

[0010] In some embodiments, the combination drug further comprises at least one pharmaceutically acceptable carrier.

[0011] In another embodiment, a kit containing combination drugs is provided.

[0012] In another embodiment, the use of combination drugs or kits in the manufacture of drugs for the prevention or treatment of a disease is provided, wherein the disease is a tumor.

[0013] In another embodiment, a method is provided for treating a subject suffering from a disease or for preventing the progression of a disease, comprising administering a combination drug or kit, wherein the disease is a tumor.

[0014] In one embodiment, the tumor is a TROP2-associated tumor.

[0015] In some embodiments, the disease is a tumor. In some embodiments, the disease includes a TROP2-positive tumor. In some embodiments, the disease includes tumors that overexpress TROP2 or have 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, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, 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 carcinoma.

[0016] In another aspect, there is provided a method for treating a subject suffering from cancer or reducing the likelihood of cancer progression, the method comprising administering to the subject an effective amount of the conjugate and administering to the subject an effective amount of an anti-PD-1 antibody.

[0017] In another aspect, there is provided the use of an effective amount of a conjugate for the manufacture of a medicament for treating a subject having cancer, the conjugate being used in combination with an effective amount of an anti-PD-1 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [Figure 1.1] Shows antibody internalization in MBA-MD-468. [Figure 1.2] Shows binding activity in Trop2 ECD. [Figure 1.3] Shows internalization in NCI-N87. [Figure 2] Shows the effect of ADC-7 and DS1062a on the viability of FaDu cells. [Figure 3] This study demonstrates the effects of ADC-1 and DS1062a on the survival rate of human pancreatic cancer cells BxPC-3. [Figure 4] This study demonstrates the effects of ADC-1 and DS1062a on the survival rate of human breast cancer cells MDA-MB-468. [Figure 5] This study demonstrates the effects of ADC-7 and DS1062a on the survival rate of gastric cancer cells NCI-N87. [Figure 6] This study demonstrates the effects of ADC-2, ADC-3, and ADC-1 on the survival rate of pharyngeal squamous cell carcinoma (FaDu). [Figure 7.1] This study demonstrates the effects of ADC-2, ADC-3, and ADC-1 on the proliferation of human pancreatic cancer cells BxPC-3. [Figure 7.2] This shows the effect of ADC-2 on the proliferation of BxPC-3. [Figure 7.3] This shows the effect of ADC-2 on FaDu proliferation. [Figure 7.4] This study demonstrates the effect of ADC-2 on the proliferation of NCI-N87. [Figure 8] This study demonstrates the inhibitory effect of ADC-1 on xenograft tumors in BxPC-3 mice. [Figure 9] This study demonstrates the inhibitory effect of ADC-1 on xenograft tumors in NCI-N87 mice. [Figure 10] The inhibitory effect of ADC-1 on xenograft tumors in BR-05-0028 mice was demonstrated. [Figure 11] This study demonstrates the inhibitory effects of ADC-2 and ADC-3 on xenograft tumors in BxPC-3 mice. [Figure 12] This study demonstrates the inhibitory effects of ADC-2, ADC-3, and ADC-1 on xenograft tumors in NCI-N87 mice. [Figure 13.1] This study demonstrates the inhibitory effects of ADC-2, ADC-3, and ADC-1 on xenograft tumors in FaDu mice. [Figure 13.2] This study demonstrates the inhibitory effect of ADC-2 on xenograft tumors in MDA-MB-468 mice. [Figure 14]The inhibitory effects of ADC-5 and ADC-6 on xenograft tumors in BxPC-3 mice were demonstrated. [Figure 15] This study demonstrates the inhibitory effects of ADC-5 and ADC-6 on xenograft tumors in NCI-N87 mice. [Figure 16] This study demonstrates the inhibitory effects of ADC-5 and ADC-6 on xenograft tumors in FaDu mice. [Figure 17] The results for serum stability of ADC-1 are shown. [Figure 18] This shows the combination of ADC2 and anti-mPD-1 in the MC38-hTROP2 syngeneic CDX model of colon cancer. [Modes for carrying out the invention]

[0019] Detailed explanation Specific embodiments are provided below to illustrate the technical content of this disclosure. Those skilled in the art will readily understand other advantages and effects of this disclosure through the content disclosed herein. This disclosure may also be implemented or applied through other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of this disclosure.

[0020] definition Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The term "technologies" as used herein refers to technologies commonly understood in the art, including obvious modifications and equivalent substitutions to those skilled in the art. The following terms are considered readily understandable to those skilled in the art, but their definitions are provided for the greater clarity of this disclosure. Where a trade name is present herein, it refers to the corresponding product or its active ingredient. All patents, published patent applications, and publications cited herein are incorporated herein by reference in their entirety for all purposes.

[0021] Where a particular quantity, concentration, or other value or parameter is expressed in the form of a range, preferred range, preferred upper limit, or preferred lower limit, it should be understood that this is equivalent to specifically revealing any range formed by combining any upper limit or preferred value with any lower limit or preferred value (whether such range is explicitly enumerated or not). Unless otherwise specified, numerical ranges enumerated herein are intended to include the endpoints of the range, as well as all integers and fractions (decimals) within that range. For example, the expression "i is an integer between 1 and 20" means that i is any integer between 1 and 20, for example, i could be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions, such as j, k, and g, should be understood in the same way.

[0022] Unless otherwise explicitly indicated by context, the singular forms "a" and "the" include the plural forms. The expressions "one or more" or "at least one" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0023] When used in relation to numerical variables, the terms "approximately" and "about" generally mean that the value of that variable, and all values ​​of that variable, are within the experimental error (e.g., within a 95% confidence interval for the mean), or within ±10% of a specified value, or a wider range.

[0024] The term "stoichiometric ratio" means that various substances are compatible according to a specific weight ratio. For example, in this disclosure, the active ingredient is mixed with an injector, a binder, and a lubricant in a specified weight ratio.

[0025] The terms “optional” or “optional” mean that the event described following the term may not necessarily occur, and such description includes whether or not such event or situation may occur.

[0026] The expressions “comprising,” “including,” “containing,” and “having” are open-ended and do not exclude additional unlisted elements, steps, or components. The expression “consisting of” excludes any unspecified elements, steps, or components. The expression “essentially consisting of” means that the scope is limited to elements, steps, or components that exist at the discretion of the specified elements, steps, or components, in addition to those that 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.”

[0027] The term "antibody," as used herein, is used broadly and includes, in particular, intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (only if they possess the desired biological activity). Antibodies may be any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and may originate from any suitable species. In some embodiments, antibodies are of human or mouse origin. Antibodies may also be fully human antibodies, humanized antibodies, or chimeric antibodies prepared by recombinant methods.

[0028] The term "monoclonal antibody" is used herein to refer to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting that population are identical except for a few possible native variations. Monoclonal antibodies exhibit high specificity for a single antigen site. The term "monoclonal" indicates that the antibody characteristics are obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring any specific antibody production method.

[0029] Intact or full-length antibodies essentially contain antigen-binding variable regions (CLs) and light chain constant regions (CHs) (which may include CH1, CH2, CH3, and CH4 depending on the antibody subtype). The antigen-binding variable region (also known as a fragment variable region or Fv fragment) typically includes a light chain variable region (VL) and a heavy chain variable region (VH). The constant regions may be constant regions with a native sequence (e.g., a constant region with a human native sequence) or amino acid sequence variants thereof. The variable regions recognize and interact with target antigens. The constant regions may be recognized and interact with by the immune system.

[0030] An "antibody fragment" may include a portion of an intact antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fd fragments, Fv fragments, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs), consisting of Fab, Fab', F(ab')2, VH, and CH1 domains. A Fab fragment is an antibody fragment obtained by papain digestion of full-length immunoglobulin, or a fragment having the same structure produced, for example, by recombinant expression. A Fab fragment includes a light chain (including VL and CL) and another chain, the other chain containing the variable domain (VH) and constant region domain (CH1) of the heavy chain. An F(ab')2 fragment is an antibody fragment obtained by pepsin digestion (pH 4.0-4.5) of immunoglobulin, or a fragment having the same structure produced, for example, by recombinant expression. An F(ab')2 fragment essentially consists of two Fab fragments, with each heavy chain portion containing several additional amino acids, including a cysteine ​​that forms a disulfide bond connecting the two fragments. A Fab' fragment is a fragment containing half of an F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments may contain multiple chains linked together, for example, via disulfide bonds and / or via peptide linkers. Examples of antibody fragments include single-chain Fv(scFv), Fv, dsFv, diabody, Fd and Fd' fragments, as well as other fragments including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and usually at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into the antibody framework (e.g., by substitution of a corresponding region), can result in an antibody that binds immunospecifically to an antigen.

[0031] The antibodies described herein 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 modified recombinant antibodies (or antibody mimes) can be expressed in a suitable culture system (e.g., Escherichia coli or mammalian cells). This manipulation may refer, for example, to the introduction of ligase-specific recognition sequences at their terminals.

[0032] Cytotoxins refer to substances that inhibit or prevent cell expression activity, cell function, and / or cause cell destruction. Cytotoxins currently used in ADCs are more toxic than chemotherapeutic drugs. Examples of cytotoxins include, but are not limited to, drugs that target the following: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and the regulation of apoptosis. Drugs that target the microtubule cytoskeleton may 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, mytansinoids, auristatin, vinblastine, colchicine, and drastatin. DNA targeting drugs may be, for example, drugs that directly disrupt DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt DNA structure include, but are not limited to, DNA double-strand cleavers, DNA alkylating agents, and DNA intercalators. DNA double-strand cleavage agents may be engine antibiotics, including but not limited to dynemycin, esperamicin, neocarlutinostatin, and unciaramycin. DNA alkylating agents may be, for example, DNA bisalkylating agents (i.e., DNA crosslinkers) or DNA monoalkylating 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-dihydrogen-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, dihydroindolebenzodiazepine (IGN) dimers, and duocalmycin-like compounds. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2), whose structures are shown below), camptothecin, and anthracyclines. RNA targeting drugs may include, for example, drugs that inhibit splicing, and examples include, but are not limited to, prazienolides.Drugs that target kinesin-mediated protein transport may include, for example, mitotic kinesin inhibitors, including but not limited to kinesin spindle protein (KSP) inhibitors.

[0033] A spacer is a structure located between different structural modules that can spatially separate them. 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 acid and non-amino acid structures, and non-amino acid structures may 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 include, but are not limited to, single amino acid sequences, sequences containing multiple amino acids, for example, sequences containing two amino acids such as GA, or for example, GGGGS (SEQ ID NO: 42), GGGGSGGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGS (SEQ ID NO: 44), etc. A self-destructing spacer is a covalent assembly tuned to correlate the cleavage of two chemical bonds after activation of a protective moiety in the precursor: upon stimulation, the protective moiety (e.g., a cleavable sequence) is removed, triggering a series of degradation reactions that sequentially release smaller molecules over time. Examples of self-destructing spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0034] The term "alkyl" refers to a linear or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which are bonded to the rest of the molecule via single bonds. Alkyl groups can contain 1 to 20 carbon atoms, C1-C 20Alkyl groups refer to groups such as C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl groups, C3 alkyl groups, C4 alkyl groups, and C3-C6 alkyl groups. Non-limiting examples of alkyl groups include, but are not limited to, 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 their isomers. A divalent radical refers to a group obtained from a corresponding monovalent radical by removing one hydrogen atom from a carbon atom with free valence electrons (may include multiple). A divalent radical has two bonding sites attached to the rest 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-), and pentylene (-C5H 10 -), hexylene (-C6H 12 This includes, but is not limited to, 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, and ethylpropylene.

[0035] As used herein, when a group is combined with another group, the bond between the groups may be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination may 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 Alkilen, C 4~10 Cycloalkylene, C4~10 When two or more divalent groups selected from heterocyclylene and -(CO)- combine to form a combination, the two or more divalent groups are linked to each other in a linear bond, 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” It can form -(CO)- and other divalent structures. The resulting divalent structure can further bond to other parts of the molecule.

[0036] The terms "antibody-conjugate drug" and "antibody-drug conjugate" have the same meaning as used herein.

[0037] Compound of formula (I) In one aspect, equation (I): The compound TIFF2026515759000012.tif32165 is provided. During the ceremony, W is hydrogen, LKb, or -C2H4-(PEG) t It is -(CO)NH2, Y is either hydrogen or LKa-LKb. However, W and Y are not hydrogen at the same time. Each LKa is independent, Selected from TIFF2026515759000013.tif27165, opSu is TIFF2026515759000014.tif32165 or a mixture thereof, Each LKb is independent, L 2 ―L 1 —B is, Each B independently forms the terminal group R 10 Either or the following: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 One of the divalent groups selected from heterocyclylene and -(CO)-, or a combination of two or more of the aforementioned divalent groups, and 3) terminal group R 10 And, R 10 This is a group that can be eliminated when it reacts with hydrogen or a group in the payload. Each L 1 This is independently a 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. Each L 2 These can be independent, combined, or C 2~20 It is an alkylene, where one or more -CH2- structures in the alkylene are -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, TIFF2026515759000015.tif4128,C 4~10 Cycloalkylene, C 4~10 It may be replaced by heterocyclylene or phenylene, where cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or halogen, -C 1~10 Alkyl, -C 1~10 Haloalkyl, -C 1~10 Alkilen-NH-R 8 and -C 1~10 Alkilen-OR 9 It is substituted with at least one substituent selected from the following: Ld2 and each Ld1 are independently bonded or -NH-C 1~20 Alkylene-(CO)-,-NH-(PEG) i -(CO)- is selected from, or is a natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or natural oligomeric amino acid is independently unsubstituted or has -(PEG) in its side chain. j -R 11 It has been replaced with, -(PEG) t -,-(PEG) i - and - (PEG) j -Each contains 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 A PEG fragment containing alkylene R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 These are, independently, hydrogen, halogen, and -C. 1~10 Alkyl, -C 1~10 Haloalkyl, C 4~10 Selected from cycloalkylenes, or R 1 and R 2 They, together with the carbon atoms to which they are bonded, form a 3-6 membered cycloalkyl group, or R 3 and R 4 Together with the carbon atoms to which they are bonded, they form a 3-6 membered cycloalkyl group. R 11 is C 1~10 It is alkyl, m is any integer between 1 and 3. n is any integer between 2 and 20. d is either 0 or any integer between 1 and 6. Each i is an independent integer between 0 and 100, preferably between 0 and 20, and preferably each i is an independent integer between 0 and 12, more preferably between 0 and 8, particularly 4. Each j is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each j is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Each t is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each t is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12.

[0038] In one embodiment, L 2 is, -(CH2) p -(CH2)2(CO)-(p is either 0 or an integer between 1 and 5), Selected from TIFF2026515759000016.tif73165 (where b is an integer between 1 and 10).

[0039] In one embodiment, p is 0 to 3, preferably 3.

[0040] In one embodiment, L 2 is -(C2H4-O) p Selected from -(CH2)2(CO)-, where p is an integer from 1 to 5, more preferably p is 2 or 4.

[0041] In one embodiment, L 2 The carbonyl group in each of the above structures is L 1 It binds to one site, while other binding sites bind to opSu.

[0042] In one embodiment, L 2 The carbonyl group in each of the above structures is L 1 It binds to one site, and the other binding sites bind to the amide. Ld2 and each Ld1 are independently either bound or The filename is TIFF2026515759000017.tif78165. Each i is an independent integer between 0 and 100. Each j and k is an independent integer between 1 and 100.

[0043] In one embodiment, each i is an independent integer between 0 and 20. In another embodiment, each i is an independent integer between 0 and 12.

[0044] In one embodiment, each j and k is an integer between 1 and 20, independently of the other. In another embodiment, each j and k is an integer between 1 and 12, independently of the other.

[0045] In one embodiment, each i is independently an integer between 0 and 8, in particular 4.

[0046] In one embodiment, each j is independently an integer between 8 and 12, in particular 8 or 12.

[0047] In one embodiment, each k is independently an integer between 1 and 7, in particular 1, 3, or 5.

[0048] In one embodiment, Ld2 and each Ld1 independently have a bond or two ends having amino and carbonyl groups, respectively. 1~20 Alkylene, or a PEG fragment of a certain length having an amino and a carbonyl group at each of its two ends (-(PEG) i (-(PEG)) or an unsubstituted PEG fragment of a certain length in the side chain. j It is one or more natural amino acids substituted with (indicated by -).

[0049] In one embodiment, -(PEG) i- is -(O-C2H4) i -or-(C2H4-O) i - and an optional additional C at one terminal 1~10 Contains alkylene, -(PEG) j - is -(O-C2H4) j -or-(C2H4-O) j - and an optional additional C at one terminal 1~10Contains alkylene. In one embodiment, -(PEG) i - is -C2H4-(O-C2H4) i -or-(C2H4-O) i Contains -C2H4-

[0050] The molecule contains two or more Ld1, B, L 2 , or L 1 If a structure exists, each Ld1, B, L 2 , or L 1 It should be understood that the structure is selected independently. The molecule has two or more R x If (x is 1, 2, 3, 4, 5, 6, 7, 8, 9 etc.) then each R x These are selected independently. In some embodiments, the "x" of the molecule (with or without an additional single apostrophe (') or multiple apostrophes (' ', ' ' ', ' ' ' ', etc.)) is, for example, R, R 1’ , R 1” , R 1’’’ , R 2’ , R 2” , R 2’’’ It is shown that in the formula, each R x (Whether or not an additional apostrophe is added is chosen independently.) 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 The same should be understood. In some embodiments, the "i" of a molecule (with or without additional numbering) is denoted, for example, i1, i2, i3, i4, etc., and the numbering does not indicate any sequence but is simply used to identify the "i". Also, each "i" (with or without additional numbering) is selected independently.

[0051] In one embodiment, the cleavable sequence 1 is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 46), Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly (SEQ ID NO: 47), Ala-Leu-Ala-Leu (SEQ ID NO: 48), Ala-Ala-Ala, and combinations thereof, and preferably, the cleavable sequence 1 is Gly-Gly-Phe-Gly.

[0052] In one embodiment, W is hydrogen.

[0053] In one embodiment, W is -C2H4-(PEG) t The formula is -(CO)NH2, where 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.

[0054] In one embodiment, R 11 C 1~6 The alkyl group is preferably methyl.

[0055] In one embodiment, n is an integer between 2 and 5, in particular 3.

[0056] In one embodiment, d is 0 or any integer from 1 to 4, preferably 0, 1, 2, or 3.

[0057] Thiosuccinimide is unstable under physiological conditions and is prone to reverse Michael addition, which leads to cleavage at the conjugation site. Furthermore, if another thiol compound is present in the system, thiosuccinimide may also undergo thiol exchange with the other thiol compound. Both of these reactions result in a reduction of the payload and toxic side effects. In this disclosure, when applied to a linker, the ring-opened succinimide structure no longer undergoes reverse Michael addition or thiol exchange, and therefore the product is more stable. The method for the ring-opening reaction can be found in WO2015165413A1.

[0058] Compounds containing the ring-opened succinimide moiety can be purified by semi-preparative / preparative HPLC or other suitable separation methods to obtain a defined composition of high purity, regardless of the efficiency of the succinimide ring-opening reaction.

[0059] The portion containing the recognition sequence of the ligase receptor or donor substrate. In one embodiment, the compound of formula (I) G n The portion is the recognition sequence of the ligase receptor substrate, which facilitates the enzymatically catalyzed coupling of the compound of formula (I) with the targeting molecule under the catalytic action of the ligase. Optionally modified targeting molecules contain the corresponding recognition sequence of the ligase receptor substrate.

[0060] 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 may, but are not limited to, those obtained by manipulating natural transpeptidases. In a preferred embodiment, the ligase is selected from the group consisting of natural saltases, non-natural saltases, and combinations thereof. Species of natural saltases include saltase A, saltase B, saltase C, saltase D, saltase L. plantarum, etc. (A detailed description can be found in US20110321183A1, which is incorporated herein by reference). The type of ligase corresponds to a ligase recognition sequence, which is used to achieve specific conjugation between different molecules or structural fragments.

[0061] In some embodiments, the ligase is a saltase selected from saltase A, saltase B, saltase C, saltase D, and saltase L. plantarum. In these embodiments, the recognition sequence of the ligase receptor substrate is selected from the group consisting of oligomeric glycine, oligomeric alanine, and oligomeric glycine / alanine mixtures having a degree of polymerization of 3 to 10. In certain embodiments, the recognition sequence of the ligase receptor substrate is G n Here, G is glycine (Gly), and n is an integer between 2 and 10.

[0062] In another specific embodiment, the ligase is saltase A derived from Staphylococcus aureus. Therefore, the ligase recognition sequence may be the enzyme's usual recognition sequence, LPXTG (SEQ ID NO: 49). In yet another specific embodiment, the ligase donor substrate recognition sequence is LPXTGJ (SEQ ID NO: 50), and the ligase receptor substrate recognition sequence is G n X can be any single amino acid, natural or non-natural, and J is an optionally labeled amino acid fragment, which 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, each amino acid independently being any natural or non-natural amino acid. In yet another embodiment, J is G m Here, m is an integer from 1 to 10. In yet another specific embodiment, the recognition sequence for the ligated donor substrate is LPETG (SEQ ID NO: 51). In yet another specific embodiment, the recognition sequence for the ligated donor substrate is LPETGG (SEQ ID NO: 52).

[0063] In one embodiment, the ligase is saltase B derived from Staphylococcus aureus, and the corresponding donor substrate recognition sequence may be NPQTN (SEQ ID NO: 53). In another embodiment, the ligase is saltase B derived from Bacillus anthracis, and the corresponding donor substrate recognition sequence may be NPKTG (SEQ ID NO: 54).

[0064] In yet another embodiment, the ligase is saltase A derived from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence may be LPXTGJ, where J is as defined above. In yet another embodiment, the ligase is saltase subfamily 5 derived from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence may be LAXTG (SEQ ID NO: 55).

[0065] In yet another embodiment, the ligase may be saltase A derived from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence may be LPQTSEQ (SEQ ID NO: 56).

[0066] Ligase recognition sequences may also be other entirely novel recognition sequences for transpeptidases optimized by manual screening.

[0067] part containing the reactive group Reactive groups involved in binding with the payload In one embodiment, B is a terminal group R 10 And L 1The cleavable sequence 1 in formula (I) binds to the payload. In such cases, B is not present in the molecule resulting from the binding of cleavable sequence 1 to 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, an ester bond, or an ether bond. In one embodiment, the reactive group in B of formula (I) is independently a reactive group for condensation reactions, nucleophilic addition or electrophilic addition (reactive C=O moiety, reactive C=CC=O moiety, amino group, amine group, hydroxyl group, or thiol group), or a reactive group for substitution reactions (e.g., a leaving group bonded to an O, C, N, or S atom). In one embodiment, the reactive group in B is independently selected from a carboxyl group, an active ester, an aldehyde group, an amino group, an amine group, a hydroxyl group, and a thiol group. In specific embodiments, the reactive group in B used for binding to the payload is independently selected from amino groups, amine groups, hydroxyl groups, thiol groups, carboxyl groups, and active esters.

[0068] In one embodiment, the reactive group in B is independently an amino group, an amine group, or a hydroxyl group, which react with the corresponding group of the payload (e.g., a carboxyl group, a sulfonic acid group, a phosphoryl group with a free -OH terminus, an active ester, an acid chloride, or an isocyanate group). In another embodiment, the reactive group in B is independently a carboxyl group or an active ester, which react with the corresponding group of the payload (e.g., an amino group, an amine group, or a hydroxyl group).

[0069] In one embodiment, the reactive group in B is independently an amino group, a hydroxyl group, or a thiol group, which reacts with the corresponding group in the payload (e.g., a halogen, a hydroxyl group, or an aldehyde group). In another embodiment, the reactive group in B is independently a hydroxyl group, which reacts with the corresponding group in the payload (e.g., a halogen or a hydroxyl group).

[0070] In one embodiment, each B is independently R 10 Either, or the following: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 3) Terminal group R 10 This is the combination of the two.

[0071] In one embodiment, Sp1 is selected from PABC, acetal, heteroacetal, and combinations thereof. In one embodiment, Sp1 is acetal, heteroacetal, or PABC. In one embodiment, heteroacetal is selected from N,O-heteroacetal. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, where -O- or -NH- is connected to cleavable array 1, and U is absent or O, S, or NH, preferably O or S. In one embodiment, U is absent or O, S, or NH, preferably O or S.

[0072] In one embodiment, B is R 10 -NH-CH2-HI 10 , -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 Or -NH-CH2-U-(CH2) g -(CO)-R 10 That is the case.

[0073] In one embodiment, R 10 is hydrogen, hydroxyl, or This is TIFF2026515759000018.tif27165. In one embodiment, R 10 is hydrogen. In one embodiment, R 10 is hydroxy or The filename is TIFF2026515759000019.tif27165.

[0074] In one embodiment, R 10 This indicates a structural portion that does not appear in the product molecule resulting from the reaction between B and the payload.

[0075] Specific Embodiments of Compound (I) In one embodiment, W is hydrogen, and each LKa is This is TIFF2026515759000020.tif27165. In one embodiment, formula (I) is formula (Ia): It has the structure of TIFF2026515759000021.tif53165.

[0076] In one embodiment, each LKa is This is TIFF2026515759000022.tif27165. In one embodiment, formula (I) is formula (Ib): It has the structure of TIFF2026515759000023.tif58165.

[0077] In one embodiment, Ld2 is a bond and d is 0. In one embodiment, the compound of formula (Ia) is as follows: As shown in TIFF2026515759000024.tif27165.

[0078] In one embodiment, d is 0, and Ld2 is The file is TIFF2026515759000025.tif27165. In one embodiment, the compound of formula (Ia) is as follows: As shown in TIFF2026515759000026.tif32165.

[0079] In one embodiment, d is 1, 2, or 3, and Ld2 and each Ld1 are independently Selected from TIFF2026515759000027.tif27165. In one embodiment, the compound of formula (Ia) is as follows: As shown in TIFF2026515759000028.tif138165.

[0080] In one embodiment, Ld2 is The filename is TIFF2026515759000029.tif42165, where d is 0. In one embodiment, the compound of formula (Ia) is as follows: As shown in TIFF2026515759000030.tif48165.

[0081] In one embodiment, d is 1, 2 or 3, and Ld2 is The filename is TIFF2026515759000031.tif42165, and each Ld1 is independent of the others. Selected from TIFF2026515759000032.tif27165. In one embodiment, the compound of formula (Ia) is as follows: As shown in TIFF2026515759000033.tif200165.

[0082] In one embodiment, d is 1, W is hydrogen, and Ld2 is The file is TIFF2026515759000034.tif42165, and each Ld1 is independent. Selected from TIFF2026515759000035.tif27165. In one embodiment, the compound of formula (Ib) is as follows: As shown in TIFF2026515759000036.tif79165.

[0083] In one embodiment, d is 1 and W is -C2H4-(PEG) t -C(O)NH2, where Ld2 is a bond, and each Ld1 independently, Selected from TIFF2026515759000037.tif27165. In one embodiment, the compound of formula (Ib) is as follows: As shown in TIFF2026515759000038.tif59165.

[0084] In one embodiment, n is 3, and L 2 is, -(CH2) p It is -(CH2)2(CO)-, p is 3, L 1 B is GGFG, and B is -NH-CH2-UR 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, Ia-0-1 is structure: It has TIFF2026515759000039.tif90165.

[0085] In one embodiment, Ia-0-2 is structure: It has TIFF2026515759000040.tif78165.

[0086] In one embodiment, Ia-0-3 is structure: It has TIFF2026515759000041.tif141165.

[0087] In one embodiment, Ia-0-4 is structure: It contains TIFF2026515759000042.tif166165 and TIFF2026515759000043.tif104165.

[0088] In one embodiment, Ia-0-5 is structure: It has TIFF2026515759000044.tif167165 and TIFF2026515759000045.tif89165.

[0089] In one embodiment, Ia-1-1 is structure: It has TIFF2026515759000046.tif136165.

[0090] In one embodiment, Ia-1-2 is structure: It has TIFF2026515759000047.tif176165.

[0091] In one embodiment, Ia-1-3 is structure: It has TIFF2026515759000048.tif238165 and TIFF2026515759000049.tif130165.

[0092] In one embodiment, Ia-1-4 is structure: It has TIFF2026515759000050.tif208165 and TIFF2026515759000051.tif109165.

[0093] In one embodiment, n is 3, and L 2 is -(C2H4-O) p It is -(CH2)2(CO)-, p is 2, L 1 B is GGFG, and B is -NH-CH2-UR 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, Ib-1 is structure: It has TIFF2026515759000052.tif181165 and TIFF2026515759000053.tif89165.

[0094] In one embodiment, n is 3, and L 2 is -(C2H4-O) p It is -(CH2)2(CO)- and p is 2, L 1 is GGFG, and B is -NH-CH2-UR 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, Ib-0 is structure: It has TIFF2026515759000054.tif202165.

[0095] In one embodiment, i is 4, g is 1, and R 11 It is methyl.

[0096] In one embodiment, i is 2, g is 1, and R 11 It is methyl.

[0097] In one embodiment, Ia-0-2 is as follows (Ia-0-2-1 to Ia-0-2-3): As shown in TIFF2026515759000055.tif101165.

[0098] In one embodiment, i is 4, j is 8, g is 1, and R 11 is methyl. In one embodiment, Ia-1-2 is as follows (Ia-1-2-1 to Ia-1-2-3): As shown in TIFF2026515759000056.tif194165.

[0099] In one embodiment, n is 3, i is 4, j is 12, g is 1, R 11 is methyl. In one embodiment, Ia-1-2 is as follows (Ia-1-2-4 to Ia-1-2-6): As shown in TIFF2026515759000057.tif199165.

[0100] In one embodiment, i is 4, j is 8, g is 1, m is 1, n is 3, R 11 is methyl. In one embodiment, Ib-1 is as follows (Ib-1-1 to Ib-1-3): As shown in TIFF2026515759000058.tif193165 and TIFF2026515759000059.tif95165.

[0101] In one embodiment, i is 4, j is 12, g is 1, m is 1, n is 3, R 11 is methyl. In one embodiment, Ib-1 is as follows (Ib-1-4 to Ib-1-1-6): As shown in TIFF2026515759000060.tif193165 and TIFF2026515759000061.tif95165.

[0102] In one embodiment, i is 4, j is 8, g is 1, m is 2, n is 3, R 11 is methyl. In one embodiment, Ib-1 is as follows (Ib-1-7 to Ib-1-9): As shown in TIFF2026515759000062.tif204165 and TIFF2026515759000063.tif100165.

[0103] In one embodiment, i is 4, j is 12, g is 1, m is 2, n is 3, R 11 is methyl. In one embodiment, Ib-1 is as follows (Ib-1-10 to Ib-1-12): As shown in TIFF2026515759000064.tif204165TIFF2026515759000065.tif100165.

[0104] In one embodiment, i is 4, t is 8, g is 1, m is 1, n is 3, R 11 is methyl. In one embodiment, Ib-0 is as follows (Ib-0-1 to Ib-0-3): As shown in TIFF2026515759000066.tif215165.

[0105] In one embodiment, i is 4, t is 12, g is 1, m is 1, n is 3, R 11 is methyl. In one embodiment, Ib-0 is as follows (Ib-0-4 to Ib-0-6): As shown in TIFF2026515759000067.tif225165.

[0106] In one embodiment, i is 4, t is 8, g is 1, m is 2, n is 3, R 11 is methyl. In one embodiment, Ib-0 is as follows (Ib-0-7 to Ib-0-9): As shown in TIFF2026515759000068.tif235165.

[0107] In one embodiment, i is 4, t is 12, g is 1, m is 2, n is 3, R 11 is methyl. In one embodiment, Ib-0 is as follows (Ib-0-10 to Ib-0-12): As shown in TIFF2026515759000069.tif235165.

[0108] Compound of formula (I) having a payload The reactive group contained in B is covalently conjugated with a payload containing another reactive group, yielding a compound of formula (I) with the payload.

[0109] In yet another embodiment, equation (II): A compound having the structure of TIFF2026515759000070.tif32165 is provided. During the ceremony, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P M is hydrogen or LKa-LKb-P. However, Q and M are not hydrogen at the same time. P is the B portion or L portion of the compound in formula (I). 1 It is a payload that is joined to a part, n, d, Ld1, Ld2, t, LKa, and LKb are defined as in equation (I).

[0110] As defined above in this specification, in the compound of formula (I), each LKb is independently L 2 ―L 1 —B, and each B independently has a terminal group R 10 Either, or the following: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 One of the divalent groups selected from heterocyclylene and -(CO)-, or a combination of two or more of the aforementioned divalent groups, and 3) terminal group R 10 And, it is a combination of R 10 R is a group that can be eliminated when it reacts with hydrogen or a group of the payload. In one embodiment, R 10 This indicates a structural portion that does not appear in the product molecule resulting from the reaction between B and the payload.

[0111] In one embodiment, P binds to the B portion of the compound of formula (I) to form the compound of formula (II). As defined above, R 10 This does not appear in the B-P structure of the compound of formula (II).

[0112] In the compound of formula (I), B is the terminal group R 10 If R 10 It should be understood that does not appear in the compound of formula (II). Therefore, as a result, B is not present in the B-P structure of the compound of formula (II).

[0113] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P, where P is the B portion or L portion of the compound of formula (I). 1 The payload is attached to the portion, and each B independently has a terminal group R 10 Either, or the following: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C4~10 One of the divalent groups selected from heterocyclylene and -(CO)-, or a combination of two or more of the aforementioned divalent groups, and 3) terminal group R 10 And, it is a combination of R 10 R is a group that can be eliminated when it reacts with hydrogen or a group in the payload. 10 This indicates a structural portion that does not appear in the product molecule resulting from the reaction between B and the payload.

[0114] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P, where each B either does not exist independently or is bonded to: 1) a self-destructing spacer Sp1, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 A combination of heterocyclylene and one or more divalent groups selected from -(CO)-. In a preferred embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P, where each B either does not exist independently, or is -NH-CH2-U-, or -NH-CH2-U-(CR 1 R 2 ) g It is -(CO)-. In another embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P. In one embodiment, LKa-L 2 ―L 1 In ―B―P, B does not exist. In one embodiment, LKa-L 2 ―L 1 In -B-P, B is: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10A combination of heterocyclylene and one or more divalent groups selected from -(CO)-. In one embodiment, LKa-L 2 ―L 1 In -B-P, B is either -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g The compound is -(CO)-, and U is either absent or O, S, or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is attached to the payload via an amide bond, an ester bond, or an ether bond.

[0115] As defined above in this specification, B in the compound of formula (I) is a terminal group R 10 In that case, B is not present in the B-P structure of the compound of formula (II). In such a case, L 1 In this case, the cleavable sequence 1 in binds to the payload to form the compound of formula (II), and it can be understood that B is not present in the molecule resulting from the binding of the cleavable sequence 1 and the payload. Therefore, in one embodiment, P is L of the compound of formula (I). 1 It binds to a portion to form the compound of formula (II). Thus, in one embodiment, M is LKa-L 2 ―L 1 ―B―P, where B does not exist, and M is LKa-L 2 ―L 1 —It can also be shown as P.

[0116] payload In this disclosure, the payload may be selected from the group consisting of small molecule compounds, nucleic acids and analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptide mimes, 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.

[0117] 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 selected from the group consisting of taxanes, mytansinoids, auristatin, eposilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinblastines, e.g., vinblastine, vincristine, vindesine, vinorelbine, vinflunin, vinglicinate, anhydrovinblastine, drastatin 10 and its analogues, halichondrin B and eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermold, and laurimaride. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, e.g., camptothecin and its derivatives, mitoxantrone, and mitogwazone. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustards, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembichin, fenamet, fenesterine, prednimustine, trophosphamide, and uracil mustard. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of nitrosoureas, e.g., carmustine, flubenzuron, formoterol, lomustine, nimustine, and ramustine. 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 benzodopa, carbocone, metsuredepa, 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 enediine antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemycin, esperamicin, neocartinostatin, and acrasinomycin.In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycin, actinomycin C, carabicin, carminomycin, and cardinophilin, carminomycin, actinomycin D, daunorubicin, detrubicin, adriamycin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, ferric adriamycin, rhodorubicin, ruhochromomycin, streptozosin, dinostatin, and zolbicin. 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, veraculin A, basilocuporin A, and anguidin. 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, azacerin, and 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folate analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of dimethyl folate, methotrexate, pteropterin, trimethrexate, 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, and 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, dideoxyuridine, doxifluridine, and 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 carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone. In another embodiment, the cytotoxin is selected from the group consisting of anti-adrenal 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, leuprorelin acetate, and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors. In yet another embodiment, the cytotoxin is selected from the group consisting of vinblastine, colchicine, taxane, auristatin, mytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, drastancine, and pyrrolobenzodiazepine (PBD). In certain embodiments, the cytotoxin is selected from the group consisting of vinblastine, colchicine, taxane, auristatin, and mytansinoids.

[0118] In certain embodiments, the cytotoxin is exatecan or a derivative thereof, such as DX8951f.

[0119] In another specific embodiment, the cytotoxin is a mytansinoid, such as DM1. It should be noted that when a cytotoxin containing a thiol moiety is used, the thiol moiety can react with the maleimide moiety to form thiosuccinimide, such as a mytansinoid, such as DM1, and the cytotoxin can be directly bound via the thiosuccinimide. In such cases, in some embodiments, the payload and the thiol moiety together constitute the cytotoxin, and therefore, in such cases, the payload represents the rest of the cytotoxin molecule excluding the thiol moiety.

[0120] In certain embodiments, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), or MMAD (monomethyl auristatin D). The synthesis and structure of auristatin compounds are described in US20060229253 (the entire disclosure of which is incorporated herein by reference).

[0121] The payload contains a reactive group that reacts with the reactive group in the compound of formula (I), and therefore allows the payload to be covalently conjugated with the compound of formula (I). Compounds that do not contain the reactive group require appropriate derivatization to obtain the payload.

[0122] In one embodiment, the cytotoxin is given by the following formula (i): It is a compound of TIFF2026515759000071.tif68165, During the ceremony, a* is either 0 or 1. The carbon atoms marked with p1* and p2* are chiral centers, and these chiral centers are in an S configuration, an R configuration, or a racemic mixture. L 1* is an unsubstituted C 1~6 Alkylene, or C substituted with one substituent selected from halogen, -OH, and -NH2 1~6 Selected from alkylenes, M* is -CH2-, -NH-, or -O-, L 2* C 1~3 It is alkylene, R 1* and R 2* These are, independently, hydrogen and C 1~6 Alkyl, halogen and C 1~6 Selected from alkoxy.

[0123] In a particular embodiment, the cytotoxin is given by the following formula (i'): It is a compound of TIFF2026515759000072.tif48165, In the formula, g is any integer between 1 and 6.

[0124] In one embodiment, g* is any integer between 1 and 3, preferably 1.

[0125] In one embodiment, L 1* C 1~6 Linear alkylene, C 1~6 Branched alkylene, C 3~6 Cyclic alkylenes and C 3~4 Cyclic alkyl-C 1~2 Selected from linear alkylene groups, each independently is either unsubstituted or substituted with one substituent selected from halogens, -OH, and -NH2. In one embodiment, L 1* is an unsubstituted C 1~4 Alkylene, or C substituted with one substituent selected from halogen, -OH, and -NH2 1~4 Selected from alkylenes. In a preferred embodiment, L 1* -CH2-, -C2H4-, Selected from TIFF2026515759000073.tif27165, each independently is either unsubstituted or substituted with at least one substituent selected from halogen, -OH, and -NH2. In a preferred embodiment, L 1* -CH2-, Selected from TIFF2026515759000074.tif27165, where "#" indicates the position of the carbonyl bond. In a more preferred embodiment, L 1* -CH2-, Selected from TIFF2026515759000075.tif22165, where "#" indicates the position of the carbonyl bond. In certain embodiments, L 1* -CH2-, Selected from TIFF2026515759000076.tif22165, where "#" indicates the position of the carbonyl bond. In preferred embodiments, the halogen is selected from F, Cl, and Br, with F being particularly important.

[0126] 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.

[0127] In one embodiment, the carbon atom marked with p1* is in an S configuration or a racemate, preferably in an S configuration. In another embodiment, the carbon atom marked with p2* is in an S configuration or a racemate, preferably in an S configuration.

[0128] In one embodiment, R 1* and R 2* These are, independently, hydrogen and C 1~3 Alkyl, halogen and C 1~3 Selected from alkoxys. In a preferred embodiment, R 1* and R 2* Each is independently selected from CH3-, F, Cl, Br, and CH3O-. In one embodiment, R 1* This is selected from CH3- and Cl. In another embodiment, R 2* It is F.

[0129] In one embodiment, a* is 0, and L 1* -CH2-, Selected from TIFF2026515759000077.tif22165, where "#" indicates the position of the carbonyl bond. In one embodiment, a* is 1, and L 1* teeth TIFF2026515759000078.tif22165, M* is O, L 2* It is -C2H4-.

[0130] In one embodiment, a* is 0, and R 1* is Cl, and R 2* F is L 1* -CH2-, Selected from TIFF2026515759000079.tif22165. In one embodiment, a* is 0, and R 1* It is CH3-, and R 2* F is L 1* teeth, Selected from TIFF2026515759000080.tif22165, in the formula, "#" indicates the position of the bond to the carbonyl group.

[0131] In one embodiment, a* is 1, and R 1* It is CH3-, and R 2* F is L 1* teeth TIFF2026515759000081.tif22165, M is O, L 2* It is -C2H4-.

[0132] In one embodiment, the cytotoxin is selected from the following compounds, where the wavy lines indicate binding sites for binding with the compound of formula (I). TIFF2026515759000082.tif218165

[0133] In some embodiments, the payload is DX8951f (compound 9), DXd-(1) (compound 10), DXd-(2) (compound 14), TIFF2026515759000083.tif49165 Preferably, DX8951f, DXd-(1), TIFF2026515759000084.tif48165more DXd-(1), TIFF2026515759000085.tif48165 Most preferred Selected from TIFF2026515759000086.tif48165.

[0134] Preparation of a compound of formula (I) having a payload In one embodiment, the bonding unit and the payload are bonded via reactive groups as defined above using any reaction known in the art, including but not limited to condensation reactions, nucleophilic additions, and electrophilic additions.

[0135] In one embodiment, the payload is a cytotoxin. In one embodiment, the binding unit-payload intermediate (numbered as LBx) is as shown in the table below. TIFF2026515759000087.tif228165TIFF2026515759000088.tif52165

[0136] Conjugates and their preparation Furthermore, a compound of formula (I) having a payload containing a portion of a ligase recognition sequence can be conjugated with other molecules containing a ligase recognition sequence, thereby being used, for example, in the preparation of targeting molecule-drug conjugates, such as antibody-drug conjugates. Thus, in yet another embodiment, a conjugate comprising a compound of formula (I), a targeting molecule, and a payload is provided.

[0137] In yet another embodiment, equation (III): A conjugate having the structure TIFF2026515759000089.tif37165 is provided. During the ceremony, n, d, Ld1, and Ld2 are as defined in equation (I), Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P M is hydrogen or LKa-LKb-P. However, Q and M are not hydrogen at the same time. P is the B portion or L portion of the compound in formula (I). 1 It is a payload that is joined to a part, A is the G of compound (I) n It is an anti-TROP2 antibody or its antigen-binding fragment that binds to a portion, where G is glycine. z is an integer between 1 and 20.

[0138] In one embodiment, LKa and LKb are as defined by formula (I).

[0139] In one embodiment, the conjugate has a drug-to-antibody ratio (DAR) of an integer or non-integer between 1 and 20.

[0140] As defined above in this specification, in one embodiment, the compound of formula (I) G n The portion is a recognition sequence of the ligase receptor substrate, thereby promoting enzymatic catalysis of the compound of formula (I) and the targeting molecule under the catalytic action of the ligase. The targeting molecule is optionally modified to include the corresponding recognition sequence of the ligase receptor or donor substrate.

[0141] An antibody (or antigen-binding fragment) reacts with the compound of formula (I) under the catalytic action of a ligase. n When conjugated with a sub-substrate, 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.

[0142] In one embodiment, the antibody (or antigen-binding fragment) comprises LPXTGJ as the recognition sequence of the ligase donor substrate, where J is as defined above. G is the corresponding recognition sequence of the ligase receptor substrate. n When conjugated with, the upstream peptide bond of glycine in the LPXTGJ sequence is cleaved by saltase A, and the resulting intermediate is G n It binds to the free N-terminus, creating a new peptide bond. The resulting sequence is LPXTG n (Sequence ID 57). Sequence G n And LPXTGJ is as defined above.

[0143] In one embodiment, P is the B portion or L portion of the compound of formula (I). 1 A is bonded to the part, and A is the G of compound (I).n A compound of formula (III) is formed by bonding to the portion.

[0144] As defined above, R 10 This does not appear in the B-P structure of the compound of formula (III). As defined above, B in the compound of formula (I) is the terminal group R 10 In this case, B is not present in the B-P structure of the compound of formula (III).

[0145] As defined above, A-G of the compound of formula (III) n In the structure, A is optional and includes a corresponding sequence resulting from the reaction between the ligase receptor substrate recognition sequence and the ligase donor substrate recognition sequence.

[0146] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P, where P is the B portion or L portion of the compound of formula (I). 1 The payload is attached to the portion, and each B independently has a terminal group R 10 Either, or the following: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 One of the divalent groups selected from heterocyclylene and -(CO)-, or a combination of two or more of the aforementioned divalent groups, and 3) terminal group R 10 And, it is a combination of R 10 R is a group that can be eliminated when it reacts with hydrogen or a group in the payload. 10 This indicates a structural portion that does not appear in the product molecule resulting from the reaction between B and the payload.

[0147] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P, where each B either does not exist independently or is bonded to: 1) a self-destructing spacer Sp1, or -CR 1 R 2 -, C1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 A combination of heterocyclylene and one or more divalent groups selected from -(CO)-. In a preferred embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P, where each B either does not exist independently, or is -NH-CH2-U-, or -NH-CH2-U-(CR 1 R 2 ) g It is -(CO)-. In another embodiment, M is hydrogen or LKa-L 2 ―L 1 —B—P. In one embodiment, LKa-L 2 ―L 1 In ―B―P, B does not exist. In one embodiment, LKa-L 2 ―L 1 In -B-P, B is: 1) self-destructing spacer Sp1, and 2) bonded, or -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 A combination of heterocyclylene and one or more divalent groups selected from -(CO)-. In one embodiment, LKa-L 2 ―L 1 In -B-P, B is either -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g The compound is -(CO)-, and U is either absent or O, S, or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is attached to the payload via an amide bond, ester bond, or ether bond. In one embodiment, M is LKa-L 2 ―L 1 ―B―P, where B does not exist, and M is LKa-L 2 ―L 1 —It can also be shown as P.

[0148] Targeting molecules In one embodiment, the targeting molecule has a heavy chain variable region (V H ) and light chain variable region (V L ) is an anti-Trop2 antibody or its antigen-binding fragment, V H teeth, (i) HCDR1 containing the amino acid sequence 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 containing the amino acid sequence WINTX4X5GX6PX7YX8X9DFKG (Sequence ID 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)X 10 The amino acid sequence of GFGSSYWYFDV (SEQ ID NO: 3) is included, where X 10 HCDR3 is G or S Includes, and / or, V L teeth, (i) LCDR1 containing the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14), (ii) LCDR2 containing the amino acid sequence of SASYRYT (SEQ ID NO: 15), (iii) LCDR3 containing the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) Includes.

[0149] In one embodiment, HCDR1 includes the amino acid sequence of NYGMN (SEQ ID NO: 4), TAGMQ (SEQ ID NO: 5), AAGMN (SEQ ID NO: 6), or NAGMN (SEQ ID NO: 7).

[0150] In one embodiment, HCDR2 contains the amino acids WINTYTGEPTYTDDFKG (SEQ ID NO: 8), WINTHSGVPKYAEDFKG (SEQ ID NO: 9), and WINTDSGEPTYTDDFKG (SEQ ID NO: 10).

[0151] In one embodiment, HCDR3 contains the amino acids GGFGSSYWYFDV (SEQ ID NO: 11) or SGFGSSYWYFDV (SEQ ID NO: 12).

[0152] In one embodiment, the antibody or its antigen-binding fragment has a heavy chain variable region (V H ) and light chain variable region (V L ) including, V H teeth, (i) HCDR1 containing the amino acid sequence X1AGMN, where X1 is N or A, (ii) HCDR2 containing the amino acid sequence WINTDSGEPTYTDDFKG (SEQ ID NO: 10), (iii) HCDR3 containing the amino acid sequence GGFGSSYWYFDV (SEQ ID NO: 11) Includes, and / or, V L teeth, (i) LCDR1 containing the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14), (ii) LCDR2 containing the amino acid sequence of SASYRYT (SEQ ID NO: 15), (iii) LCDR3 containing the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) Includes.

[0153] In one embodiment, V H teeth, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, (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 Includes, and / or, V L teeth, (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 Includes.

[0154] In one embodiment, V H teeth, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 5, (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 9, and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 12 Includes, and / or, V L teeth, (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 14, (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 Includes.

[0155] In one embodiment, V H teeth, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, (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 Includes, and / or, V L teeth, (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 Includes.

[0156] In one embodiment, V H teeth, (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 Includes, and / or, V L teeth, (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 Includes.

[0157] In one embodiment, V H teeth, (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 Includes, and / or, V L teeth, (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 Includes.

[0158] In one embodiment, V H It has the structure: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4, where FR1 contains the amino acid of SEQ ID NO: 17, FR2 contains the amino acid of SEQ ID NO: 18, FR3 contains the amino acid of SEQ ID NO: 19, and FR4 contains the amino acid of SEQ ID NO: 20.

[0159] In one embodiment, V HThis includes an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs. 21-25. In one embodiment, V H This includes the amino acid sequence of SEQ ID NO: 21. In one embodiment, V H This includes the amino acid sequence of SEQ ID NO: 22. In one embodiment, V H This includes the amino acid sequence of SEQ ID NO: 23. In one embodiment, V H This includes the amino acid sequence of SEQ ID NO: 24. In one embodiment, V H This contains the amino acid sequence of SEQ ID NO: 25.

[0160] In one embodiment, V L This includes an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27. In one embodiment, V L This includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V L This contains the amino acid sequence of SEQ ID NO: 27.

[0161] In one embodiment, V H It contains an amino acid sequence that has at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and / or V L This includes an amino acid sequence that has at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

[0162] In one embodiment, V H It contains the amino acid sequence of SEQ ID NO: 21, V L This includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V H This contains the amino acid sequence of SEQ ID NO: 22, V L This includes the amino acid sequence of SEQ ID NO: 27. In one embodiment, V H It contains the amino acid sequence of SEQ ID NO: 23, V L This includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V H It contains the amino acid sequence of SEQ ID NO: 24, V LThis includes the amino acid sequence of SEQ ID NO: 26. In one embodiment, V H This contains the amino acid sequence of SEQ ID NO: 25, V L This contains the amino acid sequence of SEQ ID NO: 26.

[0163] In one embodiment, the antibody or antigen-binding fragment includes a heavy chain constant domain (CH) containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 28, and / or a light chain constant domain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 29.

[0164] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant domain (CH) containing the amino acid sequence of SEQ ID NO: 28 and a light chain constant domain containing the amino acid sequence of SEQ ID NO: 29.

[0165] In one embodiment, the antibody or antigen-binding fragment has an equilibrium dissociation constant (K) of about 0.5 nM to about 20 nM. D ) is coupled to TROP2. In one embodiment, K D The value of is approximately 0.5nM, approximately 1nM, approximately 2nM, approximately 3nM, approximately 4nM, approximately 5nM, approximately 6nM, approximately 7nM, approximately 8nM, approximately 9nM, approximately 10nM, approximately 11nM, approximately 12nM, approximately 13nM, approximately 15nM, approximately 18nM, approximately 20nM, or / or a range between any two values ​​(including the values ​​at both ends). In one embodiment, K D The value is approximately 7.5 nM to 13.5 nM.

[0166] In one embodiment, the targeting molecule is an anti-human TROP2 antibody or its antigen-binding fragment.

[0167] In one embodiment, the antibody is a recombinant antibody selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and antibody mimics. In one embodiment, the antibody mimic is selected from scFv, minibodies, diabodies, and nanobodies.

[0168] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs. 30-33, and / or a light chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NOs. 34 or SEQ ID NOs. 35.

[0169] 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 values ​​at both ends).

[0170] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequences of SEQ ID NOs. 30-33 and / or a light chain containing the amino acid sequences of SEQ ID NOs. 34 or 35.

[0171] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 30 and a light chain containing the amino acid sequence of SEQ ID NO: 34.

[0172] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 31 and a light chain containing the amino acid sequence of SEQ ID NO: 34.

[0173] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 32 and a light chain containing the amino acid sequence of SEQ ID NO: 34.

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

[0175] Examples of anti-human TROP2 antibodies include, but are not limited to, the antibody against Trodelvi (hRS7) and the antibody against DS1062 (datopotamab). The sequences of the antibodies are shown in Table 1.

[0176] In one embodiment, the combination drug comprises an antibody or antigen-binding fragment.

[0177] (Table 1) Antibody sequences TIFF2026515759000090.tif119158TIFF2026515759000091.tif245159TIFF2026515759000092.tif238158TIFF2026515759000093.tif46158

[0178] Linker-payload and conjugated antibody With regard to conjugation with the compound of formula (I), the antibody of this disclosure may include a modified portion for binding to the Gn of the compound of formula (I). The location of such modified portion is not limited and may, for example, be located at the C-terminus or N-terminus of the heavy or light chain of the antibody.

[0179] In one embodiment, the conjugate of the present disclosure, formed by the conjugation of an anti-human TROP2 antibody with a payload, can specifically bind to TROP2 on the surface of tumor cells and selectively kill TROP2-expressing tumor cells. In another preferred embodiment, the use of the conjugate of the present disclosure or the combination pharmacopoeia of the present disclosure is provided in the manufacture of a drug 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 carcinoma, lung cancer, liver cancer, endometrial cancer, head and neck cancer, ovarian cancer, and the like.

[0180] In alternative embodiments, the modification portion for conjugation with Gn in the compound of formula (I) may be introduced, for example, at a non-terminal position of the antibody's heavy or light chain using a chemical modification method.

[0181] In one embodiment, the targeting molecule of the present disclosure is an antibody or its antigen-binding fragment, which may include terminal modifications. Terminal modifications refer to modifications at the C-terminus or N-terminus of the heavy or light chain of the antibody, which include, for example, a ligase recognition sequence. In another embodiment, the terminal modification may further include a spacer Sp2 containing 2 to 100 amino acids, with the antibody, Sp2, and ligase recognition sequence being sequentially bound. In a preferred embodiment, Sp2 is a spacer sequence containing 2 to 20 amino acids. In a particular embodiment, Sp2 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, particularly GA.

[0182] In preferred embodiments, the light chain of the antibody or its antigen-binding fragment may be of three types: wild-type (LC), a C-terminally modified light chain (LCCT) modified by direct introduction of the ligase recognition sequence LPXTG, and a C-terminally modified light chain (LCCT) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. L ) are included. The heavy chain of the antibody or its antigen-binding fragment has 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) modified by introduction of a short peptide spacer and the ligase donor substrate recognition sequence LPXTG. L ) is included. X can be any natural or non-natural single amino acid. In one embodiment, X is glycine. The sequences of the modified antibodies are shown in Table 2.

[0183] The conjugate of this disclosure may further include a payload, as described above.

[0184] In one embodiment, the combination drug includes an antibody-drug conjugate (ADC).

[0185] (Table 2) Sequence of modified antibodies TIFF2026515759000094.tif193156TIFF2026515759000095.tif142156

[0186] Specific implementations of conjugate In one embodiment, Q is hydrogen, and each LKa is This is TIFF2026515759000096.tif27165. In one embodiment, equation (III) is equation (III-a): It has the structure of TIFF2026515759000097.tif58165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0187] In one embodiment, Ld2 is a bond and d is 0. In one embodiment, the compound of formula (III-a) is as follows: As shown in TIFF2026515759000098.tif38165.

[0188] In one embodiment, d is 0, and Ld2 is The file is TIFF2026515759000099.tif27165. In one embodiment, the compound of formula (III-a) is as follows: As shown in TIFF2026515759000100.tif38165.

[0189] In one embodiment, d is 1, 2, or 3, and Ld2 and each Ld1 are independently, Selected from TIFF2026515759000101.tif27165. In one embodiment, the compound of formula (III-a) is as follows: As shown in TIFF2026515759000102.tif154165.

[0190] In one embodiment, Ld2 is The filename is TIFF2026515759000103.tif42165, where d is 0. In one embodiment, the compound of formula (III-a) is as follows: As shown in TIFF2026515759000104.tif54165.

[0191] In one embodiment, d is 1, 2 or 3, and Ld2 is The filename is TIFF2026515759000105.tif42165, and each Ld1 is independent of the others. Selected from TIFF2026515759000106.tif27165. In one embodiment, the compound of formula (III-a) is as follows: As per TIFF2026515759000107.tif199165.

[0192] In one embodiment, z is 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, and 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.

[0193] In one embodiment, LKa is This is TIFF2026515759000108.tif27165. In one embodiment, equation (III) is (III-b): It has the structure of TIFF2026515759000109.tif68165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0194] In one embodiment, Q is hydrogen, d is 1, and Ld2 is The filename is TIFF2026515759000110.tif42165, and Ld1 is, Selected from TIFF2026515759000111.tif27165. In one embodiment, the compound of formula (III-b) is as follows: As shown in TIFF2026515759000112.tif84165.

[0195] In one embodiment, Q is -C2H4-(PEG) t It is -(CO)NH2, where d is 1, Ld2 is a bond, and Ld1 is Selected from TIFF2026515759000113.tif27165. In one embodiment, the compound of formula (III-b) is as follows: As shown in TIFF2026515759000114.tif64165.

[0196] In one embodiment, z is 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.

[0197] In one embodiment, B in the compound of formula (I) is the terminal group R 10 And L 1 The cleavable sequence 1 binds to the payload to form the compound of formula (II), in which case B is absent in the molecule resulting from the binding of cleavable sequence 1 and the payload. In such a case, M is LKa-L 2 ―L 1 —B—P, and it can be understood that B does not exist in the equation. In such a case, M is also LKa-L 2 ―L 1 —It can also be represented as P. In one embodiment, n is 3 and L 2 is, -(CH2) p It is -(CH2)2(CO)-, p is 3, L 1 is GGFG, and B is -NH-CH2-U-, or absent, or -NH-CH2-U-(CR 1 R 2 ) g It is -(CO)-, U is O, and g is 1.

[0198] In one embodiment, conjugate III-a-0-1 has the following structure: It has TIFF2026515759000115.tif58165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0199] In one embodiment, conjugate III-a-0-2 has the following structure: It has TIFF2026515759000116.tif53165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0200] In one embodiment, conjugate III-a-0-3 has the following structure: It has TIFF2026515759000117.tif63165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0201] In one embodiment, conjugate III-a-0-4 has the following structure: It has TIFF2026515759000118.tif99165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0202] In one embodiment, the conjugate III-a-0-5 has the following structure: It has TIFF2026515759000119.tif84165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0203] In one embodiment, conjugate III-a-1-1 has the following structure: It has TIFF2026515759000120.tif63165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0204] In one embodiment, conjugate III-a-1-2 has the following structure: It has TIFF2026515759000121.tif187165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0205] In one embodiment, conjugate III-a-1-3 has the following structure: It has TIFF2026515759000122.tif115165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0206] In one embodiment, conjugate III-a-1-4 has the following structure: It has TIFF2026515759000123.tif99165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0207] In one embodiment, i is 4, g is 1, and R 11 It is methyl.

[0208] In one embodiment, n is 3, and L 2 is -(C2H4-O) p It is -(CH2)2(CO)-, p is 2, L1 is GGFG, B is -NH-CH2-U-, and U is O. In one embodiment, conjugate III-b-1-1 has the structure: It has TIFF2026515759000124.tif78165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0209] In one embodiment, conjugate III-a-0-2 is as follows (conjugate III-a-0-2-1): As per TIFF2026515759000125.tif54165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0210] 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): As per TIFF2026515759000126.tif69165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0211] In one embodiment, i is 4, j is 12, g is 1, and R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-1): As per TIFF2026515759000127.tif69165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0212] 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-1-2-4-2): As per TIFF2026515759000128.tif63165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0213] 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): As per TIFF2026515759000129.tif63165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0214] In one embodiment, n is 3, i is 4, j is 12, m is 1, R 11 is methyl. In one embodiment, conjugate III-b-1-1 is as follows (conjugate III-b-1-1-4): As per TIFF2026515759000130.tif73165, In the formula, A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is (Gly) n It will be modified to combine with it.

[0215] Preparation of Conjugates The conjugates of this 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, wherein the targeting molecule is modified by a ligase-recognizing sequence. The method comprises steps A and B.

[0216] Step A. Preparation of the binding unit-payload intermediate In a preferred embodiment, B of the compound of formula (I) is covalently bonded to a payload containing another reactive group via a reactive group.

[0217] The conjugation unit payload intermediates prepared using the compound of formula (I) of this disclosure have a defined structure, a defined composition, and high purity, so that when conjugation reactions with antibodies are carried out, fewer impurities are introduced, or no other impurities are introduced at all. When such intermediates are used for ligase-catalyzed site-specific conjugation with modified antibodies containing ligase recognition sequences, homogeneous ADCs with highly controllable quality are obtained.

[0218] Step B. Link the targeting molecule to the payload-containing compound (I). The targeting molecules of this disclosure can be conjugated with a compound of formula (I) (i.e., a compound of formula (II)) having a payload by any method known in the art.

[0219] Compounds of formula (I) having a targeting molecule and a payload can conjugate to each other via a substrate ligase-specific recognition sequence. 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 with a compound of formula (II) under catalytic conditions of a wild-type or optimized operational ligase or any combination thereof, and under suitable catalytic reaction conditions.

[0220] In a specific embodiment, the ligase is saltase A, and the conjugation reaction follows the scheme below: This can be shown by TIFF2026515759000131.tif22165.

[0221] The triangle represents the antibody portion, and the pentagon represents the compound portion of formula (II). n, X, and J are as defined above. G is the corresponding recognition sequence of the receptor substrate. n When conjugated with, the upstream peptide bond of glycine in the LPXTGJ sequence is cleaved by saltase A, and the resulting intermediate is G n It binds to the free N-terminus, creating a new peptide bond. The resulting amino acid sequence is LPXTG n This is the array G. n And LPXTGJ is as defined above.

[0222] Metabolism of conjugates in a physiological environment When some or all of the linkers are cleaved within tumor cells, the antitumor compound portion is released, exhibiting the antitumor effect of the antitumor compound. When the linker is cleaved at the drug-binding site, the antitumor compound is released in its inherent structure, exhibiting its inherent antitumor effect.

[0223] In one embodiment, the cleavable sequence 1 (e.g., GGFG) can be cleaved by a lysosomal enzyme (e.g., cathepsin B and / or cathepsin L).

[0224] In one embodiment, Sp1 includes a self-destructing spacer. In one embodiment, Sp1 includes a PABC, an acetal, or a heteroacetal. In one embodiment, L 1 This is GGFG. In one embodiment, the linker contains -GGFG-NH-CH2-O-. In one embodiment, -GGFG-NH-CH2-O- represents a combination of a restriction enzyme site and a self-destructing spacer, which is cleaved in the cell to release the molecule of interest (e.g., a drug).

[0225] Combination drugs and pharmaceutical preparations Another object of this disclosure is to provide a combination pharmaceutical comprising a prophylactic or therapeutic dose of the conjugate of this disclosure and an anti-PD-1 antibody, wherein the conjugate is of formula (III): It has the structure of TIFF2026515759000132.tif37165, During the ceremony, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P M is hydrogen or LKa-LKb-P. Each LKa is independent, Selected from TIFF2026515759000133.tif27165, opSu is TIFF2026515759000134.tif32165 or a mixture thereof, Each LKb is independent of L 2 ―L 1 —B is, Each B either does not exist independently, or is coupled to the following: 1) self-destructing spacer Sp1, or 2) -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 A combination of one divalent group selected from heterocyclylene and -(CO)-, or a combination of two or more of the said divalent groups, wherein B is -NH-CH2-U-, absent, or -NH-CH2-U-(CR 1 R 2 ) g It is -(CO)-, and U is either absent or O, S, or NH, preferably O or S. However, Q and M are not hydrogen at the same time. P is part B of equation (III) or L 1 It is a payload that is joined to a part, Each L 1This is independently a 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. Each L 2 These can be independent, combined, or C 2~20 It is an alkylene, where one or more -CH2- structures in the alkylene are -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, TIFF2026515759000135.tif4128,C 4~10 Cycloalkylene, C 4~10 It may be replaced by heterocyclylene or phenylene, where cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or halogen, -C 1~10 Alkyl, -C 1~10 Haloalkyl, -C 1~10 Alkilen-NH-R 8 and -C 1~10 Alkilen-OR 9 It is substituted with at least one substituent selected from the following: Ld2 and each Ld1 are independently bonded or -NH-C 1~20 Alkylene-(CO)-,-NH-(PEG) i -(CO)- is selected from, or is a natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or natural oligomeric amino acid is independently unsubstituted or has -(PEG) in its side chain. j -R 11 It has been replaced with, -(PEG)t-, -(PEG)i-, and -(PEG)j- each contain 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 A PEG fragment containing alkylene R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R7 , R 8 , R 9 These are, independently, hydrogen, halogen, and -C. 1~10 Alkyl, -C 1~10 Haloalkyl, C 4~10 Selected from cycloalkylenes, or R 1 and R 2 They, together with the carbon atoms to which they are bonded, form a 3-6 membered cycloalkyl group, or R 3 and R 4 Together with the carbon atoms to which they are bonded, they form a 3-6 membered cycloalkyl group. R 11 is C 1~10 It is alkyl, m is any integer between 1 and 3. n is any integer between 2 and 20. d is either 0 or any integer between 1 and 6. Each i is an independent integer between 0 and 100, preferably between 0 and 20, and preferably each i is an independent integer between 0 and 12, more preferably between 0 and 8, particularly 4. Each j is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each j is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Each t is independently an integer between 1 and 100, preferably between 1 and 20, preferably independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. A is an anti-TROP2 antibody or its antigen-binding fragment, preferably modified to bind to the Gn portion of formula (III), and G is glycine. z is an integer between 1 and 20.

[0226] In some embodiments, the conjugate is given by the following equation (III-a) or equation (III-b): It has the structure of TIFF2026515759000136.tif120165.

[0227] In some embodiments, the conjugate is as follows: It has the structure TIFF2026515759000137.tif211165TIFF2026515759000138.tif189165TIFF2026515759000139.tif204165, Preferably, z is 1 to 4, preferably 2. Each i, i1, i2, i3, i4 is independently an integer between 0 and 100, preferably between 0 and 20, and preferably each i, i1, i2, i3, i4 is independently an integer between 0 and 12, more preferably between 0 and 8, particularly 4. Each j is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each j is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Preferably, n is 3 and L2 is -(CH2) p It is either -(CH2)2(CO)- or -(C2H4-O) p It is -(CH2)2(CO)-, p is 2-4, L1 is Gly-Gly-Phe-Gly, and B is -NH-CH2-U-, or absent, or -NH-CH2-U-(CR 1 R 2 ) g It is -(CO)-, U does not exist, or U is O and g is 1. Each t is independently an integer between 1 and 100, preferably between 1 and 20, preferably independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. m is any integer between 1 and 3, especially 1 or 2.

[0228] In some embodiments, the payload is a cytotoxin or a fragment thereof, which may be derivatized to bind to the B or L1 portion of the compound of formula (III). Preferably, the cytotoxin is Taxanes, mytansinoids, auristatin, eposylone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinblastines, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunin, vinglycinate, anhydrovinblastine, drastatin 10 and its analogues, halichondrin B, eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermold, laurimaride, camptothecin and its derivatives, mitoxantrone, mitoggua Zon, Nitrogen Mustard, Nitrosourea, Aziridine, Benzodopa, Carbocon, Metsuredepa, Uredepa, Dynemycin, Esperamicin, Neocartinostatin, Acrasinomycin, Actinomycin, Anthramycin, Bleomycin, Actinomycin C, Carabicin, Carminomycin, Cardinophilin, Carminomycin, Actinomycin D, Daunorubicin, Detrubicin, Adriamycin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin, Nogaramycin, Olibomycin, Peplomycin, Porphyromycin, Puromycin, Ferric Adriamycin, Rhodolubicin, Ruhochromomycin, Streptozocin, Dinostatin, Zolubicin, Trichothecene, T-2 Toxin, Veracurin AA) Selected from the group consisting of basilocuporin A, anguidin, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethyl folate, methotrexate, pteropterin, trimethrexate, edatrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine, carsterone, dromostanolone propionate, epithiostanol, mepithiostan, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitors, and proteasome inhibitors, and / or Selected from and / or from vinblastine, colchicine, taxanes, auristatin, mytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, drastancin, pyrrolobenzodiazepines, exatecan, and their derivatives, and / or Auristatin, particularly selected from MMAE, MMAF, or MMAD, and / or Selected from exatecan and its derivatives, for example, DX8951f.

[0229] In some embodiments, the payload is given by equation (i): It has the structure of TIFF2026515759000140.tif68165, During the ceremony, a* is either 0 or 1. The carbon atoms marked with p1* and p2* are chiral centers, and these chiral centers are in an S configuration, an R configuration, or a racemic mixture. L 1* is an unsubstituted C 1~6 Alkylene, or C substituted with one substituent selected from halogen, -OH, and -NH2 1~6 Selected from alkylenes, M* is -CH2-, -NH-, or -O-, L 2* C 1~3 It is alkylene, R 1* and R 2* These are, independently, hydrogen and C 1~6 Alkyl, halogen and C 1~6 Selected from alkoxy.

[0230] In some embodiments, L 1* C 1~6 Linear alkylene, C 1~6 Branched alkylene, C 3~6 Cyclic alkylenes and C 3~4 Cyclic alkyl-C 1~2 Selected from linear alkylene groups, where the alkylene and cyclic alkylene are each independently unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2, preferably L 1* C 1~4 Selected from alkylenes, where the alkylene is unsubstituted or substituted with one substituent selected from halogens, -OH and -NH2, more preferably L 1* -CH2-, -C2H4-, Selected from TIFF2026515759000141.tif27165, each independently is either unsubstituted or substituted with at least one substituent selected from halogen, -OH, and -NH2, most preferably L 1* -CH2-, Selected from TIFF2026515759000142.tif22165, where "#" indicates the position of the bond to the carbonyl group.

[0231] In some embodiments, a* is 0.

[0232] R 1* C 1~6 The R1* group is selected from alkyl and halogen groups, and preferably R1* is methyl or Cl.

[0233] R 2* C 1~6 Selected from alkyl and halogen, preferably R2* is F.

[0234] In some embodiments, the payload is Selected from TIFF2026515759000143.tif218165, especially, Selected from TIFF2026515759000144.tif48165.

[0235] In some embodiments, the conjugate is Selected from TIFF2026515759000145.tif181165TIFF2026515759000146.tif207165TIFF2026515759000147.tif176165TIFF2026515759000148.tif207165TIFF2026515759000149.tif233165TIFF2026515759000150.tif212165TIFF2026515759000151.tif176165TIFF2026515759000152.tif202165, Each g is an integer between 1 and 6, preferably between 1 and 3, and more preferably between 1 and 6. Each R 1 and R 2 These are independently hydrogen, halogen, and -C 1~10 Alkyl, -C 1~10 Haloalkyl, C 4~10 Selected from cycloalkylenes, or R 1 and R 2 These, together with the carbon atoms to which they are bonded, form a 3-6 membered cycloalkyl group, preferably R 1 and R 2 It is hydrogen, Each t is independently an integer between 1 and 100, preferably between 1 and 20, preferably independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. m is any integer between 1 and 3, especially 1 or 2. z is an integer from 1 to 20, particularly 2 or 4, more preferably 2.

[0236] In some embodiments, the antibody or its antigen-binding fragment has a heavy chain variable region (V H ) and light chain variable region (V L ) including, VH is (i) HCDR1 containing the amino acid sequence 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 containing the amino acid sequence WINTX4X5GX6PX7YX8X9DFKG (Sequence ID 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)X 10 The amino acid sequence GFGSSYWYFDV (SEQ ID NO: 3) is included, where X 10 HCDR3 is G or S Includes, and / or, VL is (i) LCDR1 containing the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14), (ii) LCDR2 containing the amino acid sequence of SASYRYT (SEQ ID NO: 15), (iii) LCDR3 containing the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) Includes.

[0237] In some embodiments, V H teeth, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, (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 Includes, and / or, V L teeth, (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 Includes, or, V H teeth, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 5, (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 9, and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 12 Includes, and / or, V L teeth, (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 14, (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 Does it include, or, V H teeth, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, (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 Includes, and / or, V L teeth, (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 Does it include, or, V H teeth, (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 Includes, and / or, V L teeth, (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 Does it include, or, V H teeth, (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 Includes, and / or, V L teeth, (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 Includes.

[0238] In some embodiments, V H It contains an amino acid sequence that has at least approximately 90% sequence identity with the amino acid sequences of SEQ ID NOs. 21-25, and V L This includes an amino acid sequence that has at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

[0239] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain constant domain (CH) containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 28, and / or a light chain constant domain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 29.

[0240] In some embodiments, the antibody or antigen-binding fragment has an equilibrium dissociation constant (K) of about 0.5 nM to about 20 nM. D ) is used to connect to TROP2.

[0241] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs. 30-33, and / or a light chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs. 34 or 35.

[0242] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain C-terminal modification and / or a light chain C-terminal modification so that the antibody, Sp, and the recognition sequence of the ligated donor substrate are sequentially bound, where Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, and the recognition sequence of the ligated donor substrate is LPXTGJ, where X may be any single amino acid, whether natural or non-natural, and J may be absent or an amino acid fragment containing 1 to 10 amino acids.

[0243] In some embodiments, the modified antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NOs. 30-33 and / or the light chain of SEQ ID NOs. 40 or 41. or The modified antibody or its antigen-binding fragment contains the heavy chain of SEQ ID NOs. 36-39 and / or the light chain of SEQ ID NOs. 34 or 35.

[0244] In some embodiments, the conjugate has a drug-to-antibody ratio (DAR) of an integer or non-integer between 1 and 19.

[0245] In some embodiments, the anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody. In some embodiments, the anti-PD-1 antibody binds to human FGFR3 and / or monkey FGFR3 and / or mouse FGFR3, or the anti-PD-1 antibody binds to human FGFR3 and monkey FGFR3 but not to mouse FGFR3.

[0246] In some embodiments, the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, tripalimab, tislerizumab, cintilimab, and camrelizumab.

[0247] In some embodiments, the invention optionally further includes a pharmaceutically acceptable carrier.

[0248] The combination pharmacopoeias disclosed herein can be administered in any manner, insofar as they achieve an effect of preventing, alleviating, preventing or curing a symptom in a human or animal. For example, various suitable dosage forms, in particular injectables, such as lyophilized powders for injection, injectables, or sterile powders for injection, can be prepared according to the route of administration.

[0249] The term "medically acceptable" means that, when in contact with a patient's tissue within the bounds of ordinary medical judgment, it does not cause excessive toxicity, irritation, or allergic reactions, has a reasonable benefit-to-damage ratio, and is effective for its intended purpose.

[0250] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable 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 saline. pharmaceutically acceptable carriers also include carrier materials that bring the composition closer to physiological conditions, such as pH adjusters, buffers, and toxicity modifiers, as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. In some embodiments, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with the active ingredient for treatment. Such pharmaceutically acceptable carriers may be sterile liquids, such as water, and oils, such as petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous or glycerol solutions of glucose can also be used as liquid carriers, particularly for injection. Suitable pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers, such as acetic acid, citric acid, or phosphoric acid.

[0251] In one embodiment, the conjugate of the Disclosure has an integer or non-integer drug-to-antibody ratio (DAR) of about 1 to about 20, for example, about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, or about 1 to about 2. In a particular embodiment, the conjugate of the Disclosure has a DAR of about 2, about 4, about 6, or about 8.

[0252] A kit containing combination medications.

[0253] In some embodiments, the kit is A first packaging unit including a conjugate, A second packaging unit containing an anti-PD-1 antibody, Instructions for administering conjugates and anti-PD-1 antibodies as optional interventions are provided. Includes.

[0254] Treatment method and use Combination drugs containing a conjugate and an anti-PD-1 antibody, or kits containing a conjugate and an anti-PD-1 antibody, are useful for treating tumors and / or autoimmune diseases. Tumors sensitive to conjugate treatment include those characterized by specific tumor-associated antigens or cell surface receptors, which can be recognized by the conjugate's targeting molecules and killed by the conjugate's payload / cytotoxin.

[0255] Accordingly, in yet another embodiment, the use of a combination pharmaceutical or kit of the present disclosure is also provided in the manufacture of a drug for treating a disease, disorder or condition selected from tumors or autoimmune diseases.

[0256] In another embodiment, a combination pharmaceutical or kit of the present disclosure is provided for use in the treatment of tumors or autoimmune diseases.

[0257] In another embodiment, a combination pharmaceutical or kit of the present disclosure is provided for use in the treatment of tumors or autoimmune diseases.

[0258] In a further embodiment, a method is provided for treating a tumor or autoimmune disease, comprising administering an effective amount of the combination pharmacopoeia or kit of the present disclosure to an individual in need thereof.

[0259] In a further embodiment, a method is provided for treating a subject with cancer or for reducing the likelihood of cancer progression, comprising administering an effective amount of a conjugate having the structure of formula (III) to the subject and administering an effective amount of an anti-PD-1 antibody to the subject.

[0260] In some embodiments, the conjugate is given by formula (III): It has the structure of TIFF2026515759000153.tif37165, During the ceremony, Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P M is hydrogen or LKa-LKb-P. Each LKa is independent, Selected from TIFF2026515759000154.tif27165, opSu is TIFF2026515759000155.tif32165 or a mixture thereof, Each LKb is independent of L 2 ―L 1 —B is, Each B either does not exist independently, or is coupled to the following: 1) self-destructing spacer Sp1, or 2) -CR 1 R 2 -, C 1~10 Alkilen, C 4~10 Cycloalkylene, C 4~10 A combination of one divalent group selected from heterocyclylene and -(CO)-, or a combination of two or more of the said divalent groups, wherein B is -NH-CH2-U-, absent, or -NH-CH2-U-(CR 1 R 2 ) g It is -(CO)-, and U is either absent or O, S, or NH, preferably O or S. However, Q and M are not hydrogen at the same time. P is part B of equation (III) or L 1 It is a payload that is joined to a part, Each L 1 This is independently a 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. Each L 2 These can be independent, combined, or C 2~20 It is an alkylene, where one or more -CH2- structures in the alkylene are -CR 3 R4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, TIFF2026515759000156.tif4128,C 4~10 Cycloalkylene, C 4~10 It may be replaced by heterocyclylene or phenylene, where cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or halogen, -C 1~10 Alkyl, -C 1~10 Haloalkyl, -C 1~10 Alkilen-NH-R 8 and -C 1~10 Alkilen-OR 9 It is substituted with at least one substituent selected from the following: Ld2 and each Ld1 are independently bonded or -NH-C 1~20 Alkylene-(CO)-,-NH-(PEG) i -(CO)- is selected from, or is a natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or natural oligomeric amino acid is independently unsubstituted or has -(PEG) in its side chain. j -R 11 It has been replaced with, -(PEG) t -,-(PEG) i - and - (PEG) j -Each contains 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 A PEG fragment containing alkylene R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 These are, independently, hydrogen, halogen, and -C. 1~10 Alkyl, -C 1~10 Haloalkyl, C 4~10Selected from cycloalkylenes, or R 1 and R 2 They, together with the carbon atoms to which they are bonded, form a 3-6 membered cycloalkyl group, or R 3 and R 4 Together with the carbon atoms to which they are bonded, they form a 3-6 membered cycloalkyl group. R 11 is C 1~10 It is alkyl, m is any integer between 1 and 3. n is any integer between 2 and 20. d is either 0 or any integer between 1 and 6. Each i is an independent integer between 0 and 100, preferably between 0 and 20, and preferably each i is an independent integer between 0 and 12, more preferably between 0 and 8, particularly 4. Each j is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each j is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Each t is independently an integer between 1 and 100, preferably between 1 and 20, preferably independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. A is an anti-TROP2 antibody or its antigen-binding fragment, preferably modified to bind to the Gn portion of formula (III), and G is glycine. z is an integer between 1 and 20.

[0261] In some embodiments, the conjugate has the structure of formula (III-a) or formula (III-b) below.

[0262] In some embodiments, the conjugate is as follows: Conjugate III-a-0-1, Conjugate III-a-0-2, Conjugate III-a-0-3, Conjugate III-a-0-4, Conjugate III-a-0-5, Conjugate III-a-1-1, Conjugate III-a-1-2, Conjugate III-a-1-3, Conjugate III-a-1-4, Conjugate III-b-1-1, Conjugate III-b-0-1 It has a structure, Preferably, z is 1 to 4, preferably 2. Each i, i1, i2, i3, i4 is independently an integer between 0 and 100, preferably between 0 and 20, and preferably each i, i1, i2, i3, i4 is independently an integer between 0 and 12, more preferably between 0 and 8, particularly 4. Each j is independently an integer between 1 and 100, preferably between 1 and 20, and preferably each j is independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. Preferably, n is 3, L 2 is, -(CH2) p It is either -(CH2)2(CO)- or -(C2H4-O) p It is -(CH2)2(CO)-, p is 2-4, L1 is Gly-Gly-Phe-Gly, and B is -NH-CH2-U-, absent, or -NH-CH2-U-(CR1R2) g It is -(CO)-, U does not exist, or U is O and g is 1. Each t is independently an integer between 1 and 100, preferably between 1 and 20, preferably independently an integer between 1 and 12, more preferably between 8 and 12, particularly 8 or 12. m is any integer between 1 and 3, especially 1 or 2.

[0263] In some embodiments, the payload is a cytotoxin or a fragment thereof, and is the B portion or L portion of the compound of formula (III). 1 It may also be derivatized to bind to the portion.

[0264] In some embodiments, the cancer overexpresses TROP2 or has a TROP2 gene mutation.

[0265] In some embodiments, the cancers are breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

[0266] In some embodiments, the anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody, and / or Anti-PD-1 antibodies either bind to human FGFR3 and / or monkey FGFR3 and / or mouse FGFR3, or they bind to human FGFR3 and monkey FGFR3 but not to mouse FGFR3.

[0267] The method according to any one of claims 30 to 32, wherein in some embodiments the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, tripalimab, tislerizumab, cintilimab, and camrelizumab.

[0268] In some embodiments, the conjugate is The filename is either TIFF2026515759000157.tif58165, or the conjugate is ADC-2.

[0269] In some embodiments, the conjugate and the anti-PD-1 antibody are administered simultaneously as part of the same pharmaceutical formulation.

[0270] In some embodiments, the conjugate and the anti-PD-1 antibody are administered simultaneously as part of different pharmaceutical formulations.

[0271] In some embodiments, the conjugate and the anti-PD-1 antibody are administered at different time points.

[0272] In a further embodiment, the use of an effective amount of conjugate for the manufacture of a drug for the treatment of subjects with cancer, to be used in combination with an effective amount of anti-PD-1 antibody is provided.

[0273] In some embodiments, the cancer overexpresses TROP2 or has a TROP2 gene mutation.

[0274] In some embodiments, the cancers are breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

[0275] In some embodiments, the anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody, and / or Anti-PD-1 antibodies either bind to human FGFR3 and / or monkey FGFR3 and / or mouse FGFR3, or they bind to human FGFR3 and monkey FGFR3 but not to mouse FGFR3.

[0276] In some embodiments, the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, tripalimab, tislerizumab, cintilimab, and camrelizumab.

[0277] In some embodiments, the conjugate is The filename is either TIFF2026515759000158.tif58165, or the conjugate is ADC-2.

[0278] In some embodiments, the conjugate and the anti-PD-1 antibody are intended to be administered simultaneously as part of the same pharmaceutical formulation.

[0279] In some embodiments, the conjugate and the anti-PD-1 antibody are intended to be administered simultaneously as part of different pharmaceutical formulations.

[0280] In some embodiments, the conjugate and the anti-PD-1 antibody are intended to be administered at different time points.

[0281] 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 selectively kill TROP2-expressing tumor cells. In another preferred embodiment, the use of the conjugate (or antibody) or combination pharmacopoeia of the present disclosure in the manufacture of a drug for treating a disease, disorder, or condition selected from TROP2-positive tumors is provided. 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, colon cancer, pharyngeal squamous cell carcinoma, urothelial carcinoma, and the like.

[0282] The dosage of the conjugate (or antibody) administered to the 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 healthcare professional.

[0283] Beneficial effects The antibody-drug conjugate of the present invention, using a specially designed linker-payload, is more stable and can achieve high efficacy at a lower DAR, thus reducing side effects and increasing the therapeutic index.

[0284] This disclosure utilizes a binding unit with a unique structure to catalyze the conjugation of a targeting molecule and a payload using a ligase. The conjugate of this disclosure exhibits good homogeneity, 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.

[0285] The conjugate described herein achieves at least one of the following technical effects: (1) High inhibitory activity against target cells, or strong killing effect against target cells. (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability). (3) Favorable 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 area, etc.). (5) Advanced modular design, easy assembly of multiple drugs. [Examples]

[0286] Preparation example To more clearly explain the purpose and technical solutions, this disclosure is further described below with reference to specific examples. It should be understood that the examples are not intended to limit the scope of this disclosure. Certain experimental methods not mentioned in the following examples were carried out according to conventional experimental methods.

[0287] Instruments, materials, and reagents Unless otherwise specified, the equipment 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 7.8mm ID × 300mm, 5μm; Mobile phase: 0.2M KH2PO4, 0.25M KCl (pH 6.2); Flow rate: 0.5ml / min; Acquisition time: 30min; Injection volume: 50μl; Column temperature: 25℃; Detection wavelength: 280nm; Sample tray temperature: 8℃. 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 saltase A derived from Staphylococcus aureus is prepared in Escherichia coli.

[0288] Example 1: Construction of antibody expression vector, antibody expression, purification, and identification. 1.1 Construction of an expression vector encoding an 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 a 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 a pCDNA3.3 vector (Life Technology).

[0289] (Table 3) Antibody sequences TIFF2026515759000159.tif108165

[0290] 1.2 Expression of anti-TROP2 antibody Plasmids encoding the light and heavy chains of the anti-TROP2 antibody were paired and mixed in a 2:1 mass ratio. The plasmid pairs and PEIMAX (Polyscience) transfection reagent were separately diluted in HEK293F basic medium and then homogeneously mixed. The mixture was left at room temperature and added to HEK293F seed cell cultures. Cells were cultured at 32°C for 24 hours, sampled for analysis of cell density and viability, and replenished with 10% volume of HEK293F feed medium. The culture temperature was then changed to 32°C for the next culture. At 72 hours from incubation, the cell cultures were sampled again for analysis of cell density and viability. At 144 hours from incubation, the cell cultures were sampled again for analysis of cell density and viability.

[0291] 1.3 Purification of anti-TROP2 antibody The antibodies were 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 and 150 mM NaCl (pH 7.4). Next, the supernatant of the cell culture was obtained and applied to the column. The column was washed with 50 mM Tris and 150 mM NaCl (pH 7.4) to remove nonspecifically bound proteins. The antibodies were then 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 antibody buffer was replaced with 50 mM Tris and 150 mM NaCl (pH 7.4) using an Anicon Ultra-15 centrifugal filter (Merk Millipore).

[0292] 1.4 Binding reaction kinetics and affinity analysis Binding reaction kinetics and affinity analysis were performed. Surface plasmon resonance (SPR) analysis was performed using Sensor Chip Protein A (GE Healthcare) on a Biacore T200 (GE Healthcare) according to the manufacturer's instructions. All measurements were performed using HBS-EP. + The reaction was carried out at 25°C in a buffer solution (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 purified antibody were captured in flow cells 2 and 4 (i.e., reaction surfaces) of the sensor tip, respectively. Flow cells 1 and 3 were then subjected to HBS-EP. + It was treated with a buffer solution to function as a reference surface.

[0293] 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., analyte) (Acrobio system) were used in HBS-EP +The solution was prepared in buffer. After antibody capture, serial dilutions of TROP2 were injected at a flow rate of 30 μL / min for 3 minutes (binding phase), and buffer was flowed for 10 minutes (dissociation phase). The tip surface was regenerated by two pulses of 10 mM glycine-HCl (pH 1.5) injected at a flow rate of 50 μL / min for 30 seconds. The collected data were processed using Biacore T200 Evaluation software using methods well known in the art. The processing included the following steps: (1) setting the response to zero on the Y axis and the injection start 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 dynamical analysis using a 1:1 binding model with global fit. The results for each antibody were expressed as Ka (on-rate), Kd (off-rate), and K D This is shown as the (equilibrium dissociation constant).

[0294] 1.5 SEC-HPLC Detection of Anti-TROP2 Antibodies The antibody samples were centrifuged at 12,000 rpm for 5 minutes, and the supernatant was applied to an SEC-HPLC column to detect the proportion of monomers (corresponding to intact antibodies), high molecular weight molecules (HMW, corresponding to antibody aggregates due to aggregation), and low molecular weight molecules (LMW, corresponding to antibody fragments due to degradation) of each antibody.

[0295] TIFF2026515759000160.tif76165

[0296] Trop2 is widely expressed in normal tissues, and the affinity of Ab13 and Ab16 is reduced. Low-affinity antibodies should be able to improve safety while maintaining the efficacy demonstrated in previous experiments.

[0297] 1.6 Internalization activity MDA-MB-468 cells with good viability were trypsin-treated, collected, and suspended in cold FACS buffer (DPBS + 2% FBS). 2 × 10⁻⁶ cells were then collected. 6The cell concentration was adjusted to cells / ml. Anti-Trop2 antibody and isotype control antibody samples were fluorescently labeled by mixing them with anti-human IgG-Fc-AF647 secondary antibody in a 1:1 molar ratio at room temperature for 20 minutes. 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. Cells were incubated at 37°C for 0, 10, 30, 60, 90, 120, 180, and 240 minutes for antibody internalization, with 1 × 10⁶ cells per well at each time point. 5 The cells were transferred to a V-bottom 96-well plate. Internalization was stopped by returning the cells to an ice bath. Non-internalized cells (0 min) were divided into MAX and MIN groups, and antibody-internalized cells were marked as group I. Both MIN and group I were washed twice with quench buffer (150 mM NaCl + 100 mM glycine, pH=2.0~2.5) to dissociate the antibody surface binding. After quenching, all groups were washed twice with FACS buffer and analyzed by flow cytometry using APC channels.

[0298] The MFI data was incorporated into the following equation, and the results were analyzed using a one-phase exponential coupling function in Prism6. Isotype comparison: 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%

[0299] As shown in Figure 1, all four antibodies exhibit comparable internalization activity against MDA-MB-468.

[0300] Example 2 Preparation of the intermediate Example 2.1 Preparation of Linker-Payload 1 and Linker-Payload 2 TIFF2026515759000161.tif53165opSu is It is a mixture with TIFF2026515759000162.tif32165.

[0301] Preparation of the intermediate MC-GGFG-DXd The intermediate MC-GGFG-DXd is commercially available or prepared according to the procedure described in EP2907824. This compound is used to prepare linker-payload 1.

[0302] Preparation of Linker-Payload Intermediate 1 TIFF2026515759000163.tif63165

[0303] 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 TFA / TIS / H2O solution. The product was purified by preparative HPLC, lyophilized, and stored for use. MS m / z: [MH] - = 1382.6.

[0304] Preparation of Linker-Payload 1 Linker-payload intermediate 1 and MC-GGFG-DXd (molar ratio approximately 1:2) were weighed and dissolved in water and DMF, respectively. The mixture was then thoroughly mixed and reacted at 0–40°C for 0.5–30 hours. Once the reaction was complete, an appropriate amount of Tris base solution or another solution promoting the ring-opening reaction was directly added to the reaction mixture, and the mixture was reacted at 0–40°C for a further 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.

[0305] Preparation of Linker-Payload 2 The following linker-payload, linker-payload 2, can be prepared using the same synthesis route and reagents as linker-payload 1. TIFF2026515759000164.tif78165opSu is It is a mixture with TIFF2026515759000165.tif32165.

[0306] Example 2.2 Preparation of Linker-Payload 3 and Linker-Payload 4 Preparation of intermediate 11 TIFF2026515759000166.tif78165

[0307] Step A: N-(2-bromo-5-fluorophenyl)acetamide: To a solution of acetic anhydride (214 g, 2.10 mol) stirred in acetic acid (500 mL), concentrated H2SO4 (3 mL) was added, followed by the addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) in small amounts 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 under vacuum at room temperature to obtain 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).

[0308] Step B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) was stirred in THF (1000 mL), and n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) was added dropwise over 1 hour at -78°C. After completion, the mixture was stirred under N2 for 0.5 hours. Then, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise over 0.5 hours at -78°C, and the mixture was stirred at -78°C to room temperature for 6 hours. The mixture was poured into 500 mL of saturated NH4Cl aqueous solution at 0°C, extracted with ethyl acetate (500 mL x 3), washed with brine (250 mL x 2), dried over Na2SO4, and concentrated. The mixture was triturated in (PE / EA = 1:1, 100 mL) for 10 minutes, filtered, and the cake was collected. It was dried under vacuum to obtain 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).

[0309] Step C: N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide:N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) was stirred with CH2Cl2 (170 mL) and water (170 mL). Silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) were added to the mixture, and the mixture was stirred at 30°C for 6 hours. The mixture was filtered through Celite, washed with CH2Cl2 (100 mL), and the filtrate was concentrated and purified by FCC (EA / PE = 0-40%) to obtain 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).

[0310] Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) was stirred in THF (500 mL) at 0°C. Butyl nitrite (8.48 g, 63.28 mmol) was added, followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0°C for 2 hours. After completion, the mixture was acidified with HCl (2N) to adjust the pH to 3. The mixture was extracted with ethyl acetate (200 mL x 3), washed with brine (100 mL x 2), dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 minutes, filtered, the cake was collected, and dried under vacuum to obtain 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).

[0311] Step E: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: Add 10% Pd / C (1g) to a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (12g, 47.96 mmol) in acetic anhydride (90mL) and THF (90mL), and stir the mixture at 25°C under an H2 atmosphere for 16 hours. After cooling to 0°C, add Et3N (20mL) dropwise and stir the mixture at 0°C for 1 hour. Filter through Celite and pour the filtrate into ice water (500mL). Extract with ethyl acetate (500mL x 3), wash with brine (250mL x 2), dry over Na2SO4, and concentrate. The residue was triturated with tert-butyl methyl ether (120 mL) for 10 minutes, filtered, and the cake was collected. Drying under vacuum yielded 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).

[0312] Step F: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide: 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), aqueous HCl (2N, 150 mL) was added, and the mixture was stirred at 50°C for 7 hours. After cooling to 0°C, saturated NaHCO3 aqueous solution was added dropwise to adjust the pH to 8, extracted with ethyl acetate (200 mL x 3), washed with brine (200 mL x 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. 1H 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 MS m / z 237.1 (M+H).

[0313] 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-tetrahydronaphthalen-1,7-diyl)diacetamide (4.0 g, 16.93 mmol) in DMF (80 mL), NCS (2.26 g, 16.93 mmol) was added in small amounts at 0°C. The mixture was stirred at room temperature for 16 hours. The mixture was poured into 200 mL of ice water. A precipitate formed, which was collected by filtration and dried under vacuum at room temperature to obtain 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 H 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).

[0314] 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]indolidino[1,2-b]quinoline-1-yl)acetamide:N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene- A mixture containing 2-yl)acetamide (4.0 g, 14.78 mmol) in toluene (400 mL) was to which (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolidine-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) were added. The mixture was heated under N2 reflux for 24 hours. The solvent was removed by reduced pressure, and the mixture was purified by FCC (THF / CH2Cl2 = 0-60%) to obtain 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]indolidino[1,2-b]quinoline-1-yl)acetamide (4.1 g) as a brown solid. MS m / z 498.1 (M+H).

[0315] 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]indolidino[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]indolidino[1,2-b]quinoline-1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of concentrated HCl aqueous solution was stirred under N2 at 70°C for 36 hours. The mixture was concentrated under reduced pressure to obtain 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]indolidino[1,2-b]quinoline-10,13-dione hydrochloride (2g) as a brown solid. MS (ESI) m / z 456.1 (M+H).

[0316] Preparation of intermediate 12 (12-1, 12-2) TIFF2026515759000167.tif37165

[0317] 12-1 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]indolidino[1,2-b]quinoline-10,13-dione hydrochloride (intermediate 11) by preparative HPLC.

[0318] TIFF2026515759000168.tif157163

[0319] Preparation of Linker-Payload 3 TIFF2026515759000169.tif212165opSu is It is a mixture with TIFF2026515759000170.tif32165.

[0320] 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 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 under a nitrogen atmosphere for 5 hours. A sample was taken and the reaction was monitored by HPLC detection.

[0321] 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 to 50:100) was performed to obtain approximately 2000 mg of the target product as a white solid in 44% yield.

[0322] Preparation of compound 15 (Step B) 200 mg of compound 13 was weighed and added to a 100 ml round-bottom flask. Then, 15 ml of THF was added and the mixture was stirred to dissolve it. Next, 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 stirred overnight at room temperature. A sample was taken and the reaction was monitored by TLC (PE / EA=1:1). The starting materials had largely disappeared, and new points were detected.

[0323] The reaction was quenched by adding saturated sodium bicarbonate solution. Extraction was performed three times using 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 to 1:1) to obtain approximately 80 mg of the target product as a colorless oil in 29% yield. MS m / z: [M+H] + = 501.1

[0324] Preparation of compound 16 (Step C) 200 mg of compound 15 was weighed and added to a 100 ml 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 three times with hydrogen gas. The reaction system was kept under a hydrogen atmosphere and stirred at room temperature for 0.5 hours. A sample was taken and the reaction was monitored by TLC (DCM / MeOH = 10:1). The starting material had almost disappeared, and new points were detected.

[0325] The reaction system was filtered, and the filter cake was washed three times with EA. The filtrates were combined, concentrated to dryness, and 200 mg of the product was obtained in 100% yield as a white solid. The product can be used directly in the next reaction without purification. MS m / z: [MH] - = 409.4.

[0326] Preparation of compound 21 (Step D) Step D-1 2.0 g of dichlororesin was weighed and placed in a polypeptide synthesis tube. 10 ml of DCM was added, and the mixture was allowed to swell at room temperature for 30 minutes. The solvent was removed by vacuum aspiration. The resin was washed twice with 7 ml of DCM (each wash for 1 minute). The solvent was removed by vacuum aspiration. Next, 200 mg of compound 16 was weighed and added to a 50 ml centrifuge tube. Approximately 10 ml of DCM was added, and the solid was dissolved by shaking and added to the resin. The mixture was stirred to immerse all of the resin in the solution (if any resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). The mixture was stirred for 4-5 hours. After the reaction was complete, an appropriate amount of methanol was added. The mixture was stirred for 30 minutes. The solvent was removed by vacuum aspiration. The resins were washed sequentially with 10 mL of DMF once, then methanol once, then DMF once, then methanol once, and then DMF twice (each wash for 1 minute). The solvent was removed by vacuum aspiration. A small amount of dried resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating suitability for the next coupling step.

[0327] Step D-2 Deprotection was performed twice, each time by adding 10 mL of a pre-made 20% piperidine / DMF solution and allowing it to react for 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with 10 mL of DMF twice, then methanol once, then DMF once, then methanol once, and then DMF twice (each washing time was 1 minute). The solvent was removed by vacuum aspiration. A small amount of dried resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0328] 563 mg of Fmoc-Phe-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Then, approximately 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 the activated reaction solution.

[0329] Three molar equivalents of the activation reaction solution were added to the resin. The mixture was stirred to completely immerse the resin in the solution (if the resin adhered to the tube walls, the tube walls were washed with a small amount of DCM). Stirring was continued for 2-3 hours. After the reaction was complete, the solvent was removed by vacuum suction. The resin was washed sequentially with 10 mL of DMF twice, once with methanol, once with DMF, once with methanol, and twice with DMF (each wash for 1 minute). The solvent was removed by vacuum suction. A small amount of dried resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating suitability for the next coupling step.

[0330] Step D-3 Deprotection was performed twice, each time by adding 10 mL of a pre-made 20% piperidine / DMF solution and allowing it to react for 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with 10 mL of DMF twice, then methanol once, then DMF once, then methanol once, and then DMF twice (each washing time was 1 minute). The solvent was removed by vacuum aspiration. A small amount of dried resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0331] 531 mg of Fmoc-GG-OH and 197 mg of HOBt were added to a 50 mL centrifuge tube. Then, approximately 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIC was added and activated for 10 to 30 minutes to obtain the activated reaction solution.

[0332] Three molar equivalents of the activation reaction solution were added to the resin. The mixture was stirred to completely immerse the resin in the solution (if the resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). Stirring was continued for 2-3 hours. After the reaction was complete, the reaction solution was removed by vacuum aspiration. The resin was washed sequentially with 10 mL of DMF twice, once with methanol, once with DMF, once with methanol, and twice with DMF (each wash for 1 minute). The solvent was removed by vacuum aspiration. A small amount of dried resin was taken for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating suitability for the next coupling step.

[0333] Step D-4 Deprotection was performed twice, each time by adding 10 mL of a commercially available 20% piperidine / DMF solution and allowing it to react for 10 minutes. After the reaction was complete, the solution was removed by vacuum aspiration. The resin was washed sequentially with 10 mL of DMF twice, methanol once, DMF once, methanol once, and DMF twice (each wash for 1 minute). The solvent was removed by vacuum aspiration. A small amount of dried resin was taken 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 approximately 10 mL of DMF was added. The solid was dissolved by shaking. Next, 0.24 mL of DIEA was added to the resin. The mixture was stirred to completely immerse the resin in the solution (if the resin adhered to the tube wall, the tube wall was washed with a small amount of DCM). The mixture was stirred for 2-3 hours. After the reaction was complete, the reaction solution was removed by vacuum aspiration. The resins were washed sequentially with 10 mL of DMF twice, then with methanol once, then with DMF once, then with methanol once, and then with DMF twice (each washing time was 1 minute). The solvent was removed by vacuum aspiration. A small amount of dried resin was collected for ninhydrin detection. The resin was colorless and transparent, and the solution was yellowish, indicating suitability for the next coupling step.

[0334] Step D-5 The resin was washed twice with 10 mL of methanol. Next, the solvent was completely removed by vacuum aspiration. The resin was poured out and weighed. A lysis buffer was prepared in a 250 mL conical flask (the TFE / DCM ratio was 80% / 20%, and the volume was 7-8 times the weight of the peptide resin). The lysis buffer was added to the peptide resin and shaken thoroughly. The resin was fully immersed in the lysis buffer and dissolved at room temperature for 2-3 hours. Next, the lysis 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 lysis buffer, shaken thoroughly, and then allowed to stand for 20-30 minutes. The above system was centrifuged at 3500 rpm for 3 minutes using a 50 mL centrifuge tube, and the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under sonication, centrifuged at 3500 rpm for 3 minutes, and the supernatant was drained and discarded. The solid was placed in a centrifuge tube and air-dried overnight, then preparatively purified to obtain 125 mg of the product as a white solid in 40% yield. MS m / z: [MH] - = 641.5.

[0335] Preparation of compound 22 (Step E) 150 mg of starting compound 21 and 55 mg of TSTU were weighed and added to a 10 mL round-bottom flask. Anhydrous DMF (3 mL) was added under a nitrogen atmosphere, and the mixture was stirred for 20 minutes. Then, 18 mg of compound 12-1 and 20 μl of DIEA were sequentially added to the reaction system. The mixture was stirred under a nitrogen atmosphere at room temperature for 2 to 8 hours. Samples were taken and the reaction was monitored by HPLC. The peaks of the starting materials completely disappeared, and new peaks were detected.

[0336] The reaction system was preparatively purified, the target product was collected, and freeze-dried to obtain approximately 22 mg of the product as a yellowish solid. MS m / z: [M+H] + = 1081.0.

[0337] Preparation of linker-payload 3 (Step F) Compound 22 (30 mg) was weighed and added to a 10 ml round-bottom flask, and purified water (2 ml) was added. The mixture was stirred to dissolve the compound. 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 another solution that promotes ring-opening was directly added to the reaction mixture, and the reaction was further incubated at 0–40°C for 0.2–20 hours. The reaction was monitored by HPLC until all intermediates were consumed, and then quenched with acetic acid solution.

[0338] The reaction system was preparatively purified, the target product was collected, and freeze-dried to obtain approximately 25 mg of linker-payload 3 as a yellowish solid. MS m / z: [(M+3H) / 3] + = 1194.4.

[0339] Preparation of Linker-Payload 4 The following linker-payload 4 can be prepared using the same synthesis route and reagents as linker-payload 3. The structure of linker-payload 4 is as follows: As per TIFF2026515759000171.tif89165, opSu is It is a mixture with TIFF2026515759000172.tif32165.

[0340] Example 2.3 Preparation of Linker-Payload 5 TIFF2026515759000173.tif140165

[0341] 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 the resin was then washed several times with DMF and DCM at room temperature. The subsequent ninhydrin test showed the resin to be blue.

[0342] 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 being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction kettle along with the resin, and the reaction was stirred with nitrogen at room temperature for 2-5 hours, followed by filtration. The resin was washed sequentially with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0343] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0344] 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 being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction kettle along with the resin, and the reaction was stirred with nitrogen at room temperature for 2-4 hours, followed by filtration. The resin was washed sequentially with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0345] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0346] 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 being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction kettle along with the resin, and the reaction was stirred with nitrogen at room temperature for 2-4 hours, followed by filtration. The resin was washed sequentially with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0347] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0348] 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 being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction kettle along with the resin, and the reaction was stirred with nitrogen at room temperature for 2-4 hours, followed by filtration. The resin was washed sequentially with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0349] 2400 mL of deprotection reagent was added to completely remove Fmoc, and then the resin was washed several times with DMF and DCM at room temperature. In the subsequent ninhydrin test, the resin turned blue.

[0350] 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 being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction kettle along with the resin, and the reaction was stirred with nitrogen at room temperature for 2-4 hours, followed by filtration. The resin was washed sequentially with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow.

[0351] 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 reaction mixture was stirred at room temperature under nitrogen for 0.5 hours, after which it was filtered. This process was repeated three times, after which the resin was successively washed with DMF and DCM, and subsequently showed a blue color in the ninhydrin test.

[0352] 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 being placed in an ice bath at -10°C for 0.5 hours, the mixture was slowly added to the reaction kettle along with the resin, and the reaction was stirred with nitrogen at room temperature for 2-4 hours, followed by filtration. The resin was washed sequentially with DMF and DCM, and the subsequent ninhydrin test showed it to be colorless or pale yellow. The resin peptide was washed three times with anhydrous ethanol, filtered, and allowed to cleave.

[0353] Step 1.8 Preparation of intermediate compound b 10,000 mL of cutting reagent (TFA:TIS:H2O = 95:2.5:2.5) was added to a 10 L reactor and cooled to -10 ± 2°C. Dried and weighed resin was added. The reaction mixture was warmed to room temperature and stirred under nitrogen for 2-3 hours. The resin was then filtered and washed once with 100 mL of TFA. The filtrate and washing solution were combined.

[0354] 40 L of pre-cooled (below -10°C) cold ether was added to the product solution. The mixture was stirred for 10 minutes, 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 to 3600 rpm, the centrifugation time to 5 minutes, and the temperature of the centrifugation cavity to -5°C).

[0355] The precipitate was collected as crude compound b. The crude product was purified by preparative HPLC and freeze-dried to obtain pure compound b.

[0356] Step 2: Preparation of intermediate compound a TIFF2026515759000174.tif89165

[0357] Step 2.1 Preparation of Compound 2 Compound 1 (1 equivalent) and DMF (5 v / v) were added to the reaction flask, and the mixture was stirred under nitrogen protection to dissolve. After cooling the ice bath to 0-5°C, DIEA (3 equivalents) was added dropwise, and the mixture was stirred at 5°C for 10 minutes after the dropwise addition. Next, benzyl bromide (1.3 equivalents) was added dropwise, and after the dropwise addition was completed, the mixture was allowed to rise naturally to room temperature of approximately 20°C and stirred for 16 hours.

[0358] 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 four times with MTBE, the combined organic phase was washed with saturated brine, and then the organic phase was dried with anhydrous sodium sulfate and concentrated under vacuum to obtain a yellow crude oil, which was applied to a column by a wet method. A pale yellow oil was obtained by elution at PE / EA = 6:1 (yield was 100%).

[0359] 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 the reaction flask and stirred to dissolve. TsOH (0.1 equivalent) was weighed and added to the reactants, and the reactants were held at 20-22°C for 4 hours. The reaction solution was slowly poured into ice water and extracted three times with EA. The combined organic phase was successively washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the crude product. The product was collected by mixing the silica gel sample through a column after elution at PE / EA = 1:1, and concentrated to obtain a white solid in 40% yield.

[0360] Step 2.3 Preparation of Compound 7 Under nitrogen protection, compound 4 and DMAc (10 v / v) were added to the reaction flask and stirred to dissolve. The reaction mixture was cooled to 14-18°C, DBU (0.5 equivalent) was added dropwise, and the reaction mixture was stirred at this temperature for 1.5 hours. The completion of the reaction of the starting materials was monitored by TLC. The reaction mixture was cooled to 0-5°C, PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent), and compound 6 (0.85 equivalent) were added, and the reaction was carried out at 0-10°C for 3-4 hours. The reaction was monitored by LC-MS.

[0361] The reaction solution was added to ice water, extracted once with 2-methyltetrahydrofuran, and the aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined, washed with 0.5 M hydrochloric acid, washed with saturated NaHCO3 aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column. The product was collected by elution with DCM / MeOH and concentrated under vacuum to obtain a white solid in 78% yield.

[0362] Step 2.4 Preparation of Compound 10 Under nitrogen protection, intermediate 7 and DMAc (10 v / v) were added to the reaction flask and stirred to dissolve. The reaction mixture was cooled to 14-18°C, DBU (0.5 equivalent) was added dropwise, and the reaction mixture was stirred at this temperature for 1.5 hours. The completion of the reaction was monitored by TLC. The reaction mixture was cooled to 0-5°C, PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent), and compound 9 (0.85 equivalent) were added, and the reaction was allowed to proceed at 0-10°C for 3-4 hours. The reaction was monitored by LC-MS.

[0363] The reaction solution was added to ice water, extracted once with 2-methyltetrahydrofuran, and the aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined and washed with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column. The product was collected by elution with DCM / MeOH and concentrated under vacuum to obtain a white solid in 50% yield.

[0364] Step 2.5 Preparation of Compound A Under nitrogen protection, intermediate 10 was dissolved in DCM (15 v / v), and DBU (0.5 equivalents) 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 LC-MS. The reaction solution was diluted with DCM and purified by column chromatography using a wet method. The product was collected by elution of DCM:MeOH to obtain a white solid in 82% yield.

[0365] Step 3: Preparation of intermediate compound c TIFF2026515759000175.tif89165

[0366] Compound b (400 mg, 0.245 mmol) and compound a (377 mg, 0.539 mmol) were dissolved in DMF (6 ml), and then DIPEA (159 mg, 1.23 mmol) and HATU (233 mg, 0.613 mmol) were added to the reaction solution, and the reaction was stirred at room temperature for 2 hours. After compound b disappeared, it was purified by preparative HPLC, and the prepared solution was lyophilized to obtain 380 mg of product in 52% yield. 142 H 207 O 49 N 21 [(M+3H) / 3] + Calculated value for this: 997.8, measured value: 875.9 (fragmented mass).

[0367] Step 4: Preparation of intermediate compound d TIFF2026515759000176.tif89165

[0368] 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 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 departure of the starting material to prevent an increase in the de-Fmoc product. The reaction solution was filtered and purified by preparative HPLC to obtain 270 mg of product in 76% yield. 128 H 195 O 49 N 21 [(M+3H) / 3] + Calculated value: 937.8, measured value: 875.9 (fragmented mass).

[0369] Step 5: Preparation of intermediate compound e TIFF2026515759000177.tif99165

[0370] Compounds d (270 mg, 0.096 mmol) and 12-1 (120 mg, 0.211 mmol) were dissolved in DMF (5 ml), and then DIPEA (62 mg, 0.48 mmol) and HATU (92 mg, 0.24 mmol) were added to the reaction solution and stirred at room temperature for 2 to 16 hours. After the completion of the reaction, which was monitored by HPLC, the reaction mixture was directly purified by preparative HPLC, the collected eluents were combined, and lyophilized to obtain 235 mg of product in 66% yield. 174 H 229 O 55 Cl2F2N 27 [(M+3H) / 3] + Calculated value: 1229.2, Measured value: 1229.3.

[0371] Step 6: Preparation of Linker-Payload 5 TIFF2026515759000178.tif99165

[0372] Compound e (210 mg, 0.057 mmol) was dissolved in DMF (5 ml), then diethylamine (0.5 ml) was added, and the reaction mixture was allowed to react at room temperature for 15 minutes. The reaction endpoint was monitored by HPLC. After the reaction was complete, the mixture was neutralized with 10% TFA aqueous solution under ice bath, and the reaction mixture was purified by preparative HPLC. After lyophilization, 145 mg of the product was obtained in 73% yield. 159 H 219 O 53 Cl2F2N 27 [(M+3H) / 3] + Calculated value: 1155.2, Measured value: 1155.3.

[0373] Example 3: Preparation of Targeting Molecules-Pharmaceutical Conjugates 3.1 Preparation of DS1062a analogs and Trodelvy DS1062a analog (DS1062a and DS-1062 * ) was prepared based on the method described in patent US20160297890A or manufactured by WuXi Biologics.

[0374] The Trodelvy was available for sale.

[0375] 3.2 Preparation of ADC-1 3.2.1 Processing of GQhRS7 GQhRS7 samples were treated with ultrafiltration, dialysis, or desalting column chromatography. The storage solution was replaced with ligase buffer.

[0376] 3.2.2 Enzyme-catalyzed coupling of ADC-1 ADC-1 was prepared by a coupling reaction of GQhRS7 and linker-payload 1 under the catalytic action of wild-type saltase A or a mutant ligase optimized and manipulated therebased. The modified antibody and linker-payload were thoroughly mixed in a ligase buffer in a molar ratio of 1:1 to 1:100 and added to a solid-phase coupling system. The solid-phase coupling system contained ligase immobilized in the matrix of the solid-phase coupling system. The immobilized ligase catalyzed the coupling reaction of the antibody GQhRS7 with linker-payload 1. The coupling reaction was carried out at 4 to 40°C for 0.5 to 20 hours. After the reaction was complete, the reaction mixture was ultrafiltered or dialyzed to remove unreacted intermediates and obtain ADC-1. ADC-1 was stored at 4°C or -80°C in a buffer containing 20 mM citrate and 200 mM NaCl (pH 5.0).

[0377] 3.2.3 HIC-HPLC Detection and Analysis of ADC-1 The DAR (drug-to-antibody ratio) distribution of ADC-1 was analyzed by HIC-HPLC. The cytotoxin-free antibody GQhRS7 was present in less than 5% of the product. The coupling product mainly contained ADC-1, with a DAR of 3.5.

[0378] 3.2.4 SEC-HPLC Detection and Analysis of ADC-1 The degree of high molecular weight aggregation of ADC-1 was analyzed by SEC-HPLC. The results showed that no high molecular weight polymers were detected in ADC-1. This indicates that the coupling reaction conditions were mild and did not cause damage to the antibody structure.

[0379] 3.3 The linker-payload intermediates were each site-specifically conjugated to antibodies using ligase to form ADCs. The method for the conjugation reaction can be found in WO2015165413A1. The obtained ADCs are listed in the table below. TIFF2026515759000179.tif59165

[0380] 3.4 Binding activity Human Trop2 ECD at a concentration of 0.5 μg / mL was coated onto 96-well plates overnight at 4°C. The plates were then blocked with 3% BSA-PBST at room temperature for 1 hour. After washing with PBST (0.05% Tween), a series of test samples containing different concentrations of ADC-2 (i.e., ADC2), Trodelvy, and GQhRS7 were added to the 96-well plates and incubated at room temperature for 60 minutes. After incubation, goat anti-human FC secondary antibody (HRP) (Sinobiological, SSA001) was added in a ratio of 1:100000 and incubated again at room temperature for 60 minutes. After washing, the plates were treated with TMB solution (Sigma, T0440) as an HRP substrate, and the reaction was stopped with 1 M H2SO4. The absorbance of each well was detected at a wavelength of 450 nm.

[0381] The results of this analysis are shown in Figure 1.2. These results demonstrate that ADC-2 has similar binding affinity to GQhRS7 and Trodelvy.

[0382] 3.5 Internalization activity NCI-N87 cells with good viability were trypsin-treated, collected, and suspended in cold FACS buffer (DPBS + 2% FBS). Cells were incubated on ice for 1 hour with a final concentration of 50 μg / ml of the test drug solution. The antibody-cell mixture was washed twice with cold FACS buffer to remove excess antibody. Antibody-bound cells were fluorescently labeled by mixing with a 500-fold diluted ice-cold anti-human IgG-Fc-AF647 secondary antibody solution for 30 minutes. After fluorescent labeling, the antibody-cell mixture was washed twice again. For test drug internalization, cells were incubated at 37°C for 10, 30, 60, 90, 150, and 210 minutes. Cells were suspended in quench buffer (150 mM NaCl + 100 mM glycine (pH=2.0~2.5)) to dissociate antibodies bound to the cell surface. After acidification, the cells were washed twice with cold FACS buffer and analyzed for APC channels by flow cytometry.

[0383] The MFI data was incorporated into the following equation, and the results were analyzed in Prism8 using a one-phase exponential coupling function. Internalization amount: A = MFI(I) - MFI(MIN). Internalization rate: R = [MFI(I) - MFI(MIN)] / [MFI(MAX) - MFI(Blank)] × 100%.

[0384] As shown in Figure 1.3, ADC2 exhibits comparable internalization activity to DS1062, Trodelvy, and GQhRS7 in NCI-N87.

[0385] Effect Example 1: Bystander Killing Effect of Conjugates in BxPC-3 / HepG2 Effect Example 1.1 Bystander Killing Effect of ADC-1 against HepG2 in BxPC-3 / HepG2 Co-culture Assay The cell concentrations of Trop2-positive cells BxPC-3 and Trop2-negative cells HepG2 were measured at 1x10⁻¹⁰ 6The cells were adjusted to the required concentration per mL, and 200 μL per well (cell volume BxPC-3 : HepG2 = 4 : 1) was seeded into a 6-well plate. 2.8 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium was added to each well. The cells were incubated overnight in a cell incubator at 37°C and 5% CO2. 3 mL of 20 nM ADC-1 and DS1062a were added to the cells cultured overnight (final drug concentration was 10 nM per well). A negative control group was established: 3 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium was added to each well. After treatment, the cells were transferred to an incubator and incubated for 96 hours. After incubation, the cells were digested, washed once with 1X PBS, transferred to a flow tube, and centrifuged at 2000 rpm for 3 minutes. Next, the supernatant was discarded, and cell volume 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. 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 resulting cells were mixed and incubated at 4°C for another 30 minutes. Finally, cells were washed with 1X PBS, the supernatant was discarded after centrifugation, the cells were resuspended in PBS, detected by flow cytometry, and analyzed using FlowJo software. The results are shown in Table 4.

[0386] (Table 4) Results of bystander killing tests for ADC-1 and DS1062a TIFF2026515759000180.tif39165

[0387] Effect Example 1.2 Bystander Killing Effect of ADC-2 against HepG2 in BxPC-3 / HepG2 Co-culture Assay The cell concentrations of Trop2-positive cells BxPC-3 and Trop2-negative cells HepG2 were measured at 1x10⁻¹⁰ 6The cells were adjusted to the required cell / mL concentration, and 300 μL per well (cell volume BxPC-3 : HepG2 = 2 : 1) was seeded into a 6-well plate. The wells were then supplemented with 2.7 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium. The cells were incubated overnight in a cell incubator at 37°C and 5% CO2. 3 mL of 20 nM ADC-2 and DS1062a were added to the cells cultured overnight (final drug concentration: 10 nM per well). Furthermore, the bystander-killing effects of ADC-2 and DS1062a were evaluated using the same process as above. The results are shown in Table 5.

[0388] (Table 5) Results of bystander killing tests for ADC-2 and DS1062a TIFF2026515759000181.tif38165

[0389] Example 1.3 Bystander killing effect of ADC-6 against HepG2 in a BxPC-3 / HepG2 co-culture assay The bystander-killing effects of the control (datopotamab), DS1062a, ADC-1, ADC-2, ADC-4, and ADC-6 were evaluated using the same process as in Efficacy Example 1.2. The results are shown in Table 6.

[0390] (Table 6) Results of ADC-6 bystander killing tests TIFF2026515759000182.tif58165

[0391] conclusion The experimental results for Effect Example 1.1 showed that both ADC-1 and the DS1062a analog had bystander-killing effects, and there was no significant difference in their effectiveness.

[0392] The experimental results for Effect Example 1.2 showed that the bystander killing effect of ADC-2 was better than that of DS1062a.

[0393] The experimental results for Effect Example 1.3 showed that ADC-1, ADC-2, ADC-4, ADC-6, and the DS1062a analog all possessed bystander-killing effects. The bystander-killing effects of ADC-2 and ADC-6 were better than those of DS1062a. The bystander-killing effect of ADC-6 was significantly better than that of ADC-4.

[0394] Example of effect 2: Effects of TROP2-targeting conjugates on cell proliferation Example of effect 2.1 Inhibitory effect of ADC-7 and ADC-1 on tumor cell proliferation Example of effect 2.1.1 Inhibitory effect of ADC-7 on the proliferation of human pharyngeal squamous cell carcinoma FaDu 2000 human pharyngeal squamous cell carcinoma (FaDu) cells with high TROP2 expression were seeded into 96-well plates containing 100 μL of culture medium per well and incubated overnight in a cell incubator at 37°C and 5% CO2. 100 μL of ADC-7 and DS1062a at 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) were added to each well (three replication wells for each concentration). A positive control group was established: 100 μL of puromycin at a concentration of 10 μg / mL was added to each well. A negative control group was established: 100 μL of complete FaDu cell medium was added to each well. After administration, the cells were transferred to an incubator and incubated for 96 hours. The 96-well plate was removed from the 37°C cell incubator and allowed to equilibrate to room temperature for 30 minutes. After discarding the culture 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. The luminescence signal, which reflects cell viability, was detected using a microplate reader. The inhibitory effect of the test drug on the proliferation of human pharyngeal squamous cell carcinoma FaDu is shown in Figure 2 and Table 7.

[0395] (Table 7) Inhibitory effects of ADC-7 and DS1062a on the proliferation of pharyngeal squamous cell carcinoma FaDu TIFF2026515759000183.tif24165

[0396] Example of effect 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 from Example 2.1.1 was replaced with human pancreatic cancer cells BxPC-3 (100 μL per well containing 2000 cells), and the inhibitory effect on 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.

[0397] (Table 8) Inhibitory effects of ADC-1 and DS1062a on the proliferation of human pancreatic cancer cells BxPC-3 TIFF2026515759000184.tif24165

[0398] Example of effect 2.1.3 Inhibitory effect of ADC-1 on the proliferation of human breast cancer cells MDA-MB-468 In Example 2.1.1, human pharyngeal squamous cell carcinoma FaDu was replaced with human breast cancer cells MDA-MB-468 (100 μL per well containing 4000 cells), and the inhibitory effect on 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.

[0399] (Table 9) Inhibitory effects of ADC-1 and DS1062a on the proliferation of human breast cancer cells MDA-MB-468 TIFF2026515759000185.tif24165

[0400] Example of effect 2.1.4 Inhibitory effect of ADC-7 on the proliferation of human gastric cancer cells NCI-N87 In Example 2.1.1, human pharyngeal squamous cell carcinoma FaDu 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.

[0401] (Table 10) Inhibitory effects of ADC-7 and DS1062a on the proliferation of gastric cancer cells NCI-N87 TIFF2026515759000186.tif24165

[0402] Example of effect 2.2 Inhibitory effect of ADC-2 and ADC-3 on tumor cell proliferation Example of effect 2.2.1 Inhibitory effect 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 effect was evaluated using the same process. The inhibitory effect of the test drugs on the proliferation of human pharyngeal squamous cell carcinoma FaDu is shown in Figure 6 and Table 11.

[0403] (Table 11) Inhibitory effects of ADC-2, ADC-3, and ADC-1 on the proliferation of pharyngeal squamous cell carcinoma FaDu TIFF2026515759000187.tif30165

[0404] Example of effect 2.2.2 Inhibitory effect of ADC-2 and ADC-3 on the proliferation of human pancreatic cancer cells BxPC-3 In Example 2.2.1, human pharyngeal squamous cell carcinoma FaDu was replaced with human pancreatic cancer cells BxPC-3, and the inhibitory effect on ADC-2 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 7.1 and Table 12.

[0405] (Table 12) Inhibitory effects of ADC-2, ADC-3, and ADC-1 on the proliferation of human pancreatic cancer cells BxPC-3 TIFF2026515759000188.tif31165

[0406] 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 conjugates on tumor cell proliferation. The test drugs included the 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 various concentrations of the specified drugs for 168 hours. Cell viability was examined using the CellTiter-Glo® Luminescent Cell Viability Assay, and the percentage of viable cells was calculated.

[0407] In Trop2-positive BxPC-3, FaDu, and NCI-N87, ADC2 showed more potent cytotoxicity than DS1062a. IC of ADC2 50 The value is lower than that of DS1062a (see the table below). TIFF2026515759000189.tif46165

[0408] conclusion The results of Effect Example 2.1.1 showed that both ADC-7 and DS1062a could inhibit the proliferation of FaDu cells, with ADC-7 exhibiting a slightly better inhibitory effect.

[0409] The results of Effect Example 2.1.2 showed that both ADC-1 and DS1062a can inhibit the proliferation of BxPC-3 cells.

[0410] The results of Effect Example 2.1.3 showed that both ADC-1 and DS1062a can inhibit the proliferation of MDA-MB-468 cells.

[0411] The results of Effect Example 2.1.4 showed that both ADC-7 and DS1062a can inhibit the proliferation of NCI-N87 cells.

[0412] The results of Effect Example 2.2.1 showed that ADC-2, ADC-3, and ADC-1 can inhibit the proliferation of FaDu cells.

[0413] The results of Effect Example 2.2.2 showed that ADC-2, ADC-3, and ADC-1 could inhibit the proliferation of BxPC-3 cells, and that the inhibitory effect of ADC-2 was slightly better than that of ADC-3 and ADC-1.

[0414] The results in Effect Example 2.3 showed that ADC-2 could inhibit the proliferation of BxPC-3 cells, FaDu cells, and NCI-N87 cells, demonstrating that the inhibitory effect of ADC-2 was better than that of DS1062a.

[0415] Example of effect 3: In vivo efficacy evaluation trial Efficacy Example 3.1 Evaluation of In Vivo Efficacy of ADC-1 Efficacy Example 3.1.1 Evaluation of the in vivo efficacy of ADC-1 against BxPC-3 cells I. Collect BxPC-3 in the logarithmic growth phase and measure the cell density in Matrigel buffer (PBS:Matrigel = 1:1) at 10x10⁻¹⁰. 6 The cell concentration was adjusted to cells / mL. 0.2 mL of the prepared BxPC-3 cell suspension was subcutaneously injected into the right scapula of 6-8 week old SPF female BALB / c nude mice.

[0416] II. Measure the tumor diameter with calipers and calculate the tumor volume using the formula V = 0.5 axb 2 The calculation was performed according to the formula (where a is the longest diameter of the tumor and b is the shortest diameter). Six days after cell inoculation, the average tumor volume was approximately 151 mm². 3 At this point, the animals were randomly divided into a vehicle control group, a 3 mg / kg DS1062a group, and a 3 mg / kg ADC-1 group, with 6 animals in each group. Each group received the treatment via tail vein injection, while the control group received an equal volume of vehicle. Tumor volume was measured twice weekly within 35 days of administration, and the tumor volume at day 35 was compared between the groups. T / C and TGI values ​​were calculated using tumor volume. The formula is as follows: T / C% = T RTV / CRTV × 100% (T RTV : RTV of the treatment group, C RTV (RTV of the vehicle control group). Relative tumor volume (RTV) is calculated based on the results of tumor measurements, and the formula is RTV = V t / V0, where V0 is the mean tumor volume measured at the time of group assignment (i.e., D0), V t This is the average tumor volume in a single measurement, and T RTV and C RTV Therefore, data from the same day was used. Calculation of TGI (%): TGI (%) = [1 - (average tumor volume at the end of treatment in the treatment group - average tumor volume at the start of treatment in the treatment group) / (average tumor volume at the end of treatment in the vehicle control group - average tumor volume at the start of treatment in the vehicle control group)] × 100%.

[0417] III. After 35 days of administration, the mean tumor volume in the ADC-1 3 mg / kg group (T / C = 49.05%, TGI = 57.56%, p = 0.002) and the DS1062a 3 mg / kg group (T / C = 64.05%, TGI = 40.54%, p = 0.010) was 648 mm², both groups. 3 and 847mm 3 The results are shown in Figure 8 and Table 13.

[0418] (Table 13) Inhibitory effect of ADC-1 on xenograft tumors in BxPC-3 mice TIFF2026515759000190.tif57165

[0419] conclusion According to the results in Efficacy Example 3.1.1, both the ADC-1 3 mg / kg group and the DS1062a 3 mg / kg group were able to significantly inhibit tumor growth compared to the control group.

[0420] Efficacy Example 3.1.2 Evaluation of the in vivo efficacy of ADC-1 against NCI-N87 gastric cancer cells In Example 3.1.1, BxPC-3 cells were exchanged for NCI-N87 gastric cancer cells, and 7 days after cell inoculation, the average tumor volume was approximately 227 mm². 3At this point, the animals were randomly divided into a vehicle control group, an IMMU-132 (Trodelvy) 3 mg / kg group, a DS1062a 3 mg / kg group, and an ADC-1 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-1 was evaluated using the same process as in Efficacy Example 3.1.1. The results are shown in Figure 9 and Table 14.

[0421] (Table 14) Inhibitory effect of ADC-1 on xenograft tumors in NCI-N87 mice TIFF2026515759000191.tif87165

[0422] conclusion After 32 days of administration, the mean tumor volume in the ADC-1 3 mg / kg group (T / C = 57.17%, TGI = 55.09%, p < 0.001) and the DS1062a 3 mg / kg group (T / C = 49.98%, TGI = 60.23%, p < 0.001) was 697 mm², both in the mean tumor volume groups. 3 and 658mm 3 Both methods were able to significantly inhibit tumor growth.

[0423] Efficacy Example 3.1.3 Evaluation of in vivo efficacy of ADC-1 for human breast cancer BR-05-0028 The human breast cancer BR-05-0028 model (IHC 3+) was derived from tumor samples excised during clinical surgery. The tumor samples were inoculated into P0 generation nude mice, and the tumor tissue used in this example was from the P5 generation. Approximately 30 mm 3 Tumor tissue of a certain volume was subcutaneously inoculated into the right posterior dorsal region of 6-8 week old SPF female Balb / c nude mice. 28 days after tumor tissue inoculation, the average tumor volume was approximately 171 mm². 3 At this point, the animals were randomly divided into four groups: a vehicle control group, an IMMU-132 5 mg / kg group, a DS1062a 5 mg / kg group, and an ADC-1 5 mg / kg group, with six animals in each group. The in vivo efficacy of ADC-1 was evaluated using the same process as in Efficacy Example 3.1.1. The results are shown in Figure 10 and Table 15.

[0424] (Table 15) Inhibitory effect of ADC-1 on xenograft tumors in BR-05-0028 mice TIFF2026515759000192.tif105165

[0425] conclusion After 21 days of administration, the IMMU-132 5 mg / kg group (T / C=0.35%, TGI=111.76%, p=0.037), the DS1062a 5 mg / kg group (T / C=0.48%, TGI=111.60%, p=0.037), the ADC-1 5 mg / kg group (T / C=0.23%, TGI=111.88%, p=0.037), and the ADC-1 10 mg / kg group (T / C=0.23%, TGI=111.88%, p=0.037) were able to significantly inhibit tumor growth.

[0426] Efficacy Example 3.2 In vivo efficacy evaluation of ADC-2 and ADC-3 Example 3.2.1 Evaluation of in vivo efficacy of ADC-2 and ADC-3 against BxPC-3 cells Six days after cell inoculation, the average tumor volume was approximately 151 mm². 3 At this point, 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 of ADC-2 and ADC-3 was evaluated using the same process as in Efficacy Example 3.1.1. The results are shown in Figure 11 and Table 16.

[0427] (Table 16) Inhibitory effects of ADC-2 and ADC-3 on xenograft tumors in BxPC-3 mice TIFF2026515759000193.tif87165

[0428] conclusion After 28 days of administration, the mean tumor volume was 116 mm in both the ADC-2 3 mg / kg group (T / C=12.72%, TGI=104.54%, p=0.001) and the DS1062a 3 mg / kg group (T / C=58.49%, TGI=48.52%, p=0.049). 3and 540mm 3 The study demonstrated that the 3 mg / kg group of ADC-2 could significantly inhibit tumor growth.

[0429] Example 3.2.2 Evaluation of in vivo efficacy of ADC-2 and ADC-3 against NCI-N87 gastric cancer cells In Example 3.2.1, BxPC-3 pancreatic cancer cells were exchanged for NCI-N87 gastric cancer cells. Eight days after cell inoculation, the average tumor volume was approximately 188 mm². 3 At this point, 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 of ADC-2 was evaluated using the same process as in Efficacy Example 3.2.1. The results are shown in Figure 12 and Table 17.

[0430] (Table 17) Inhibitory effect of ADC-2 on xenograft tumors in NCI-N87 mice TIFF2026515759000194.tif81165

[0431] conclusion After 28 days of administration, the mean tumor volumes were 89, 212, 293, and 255 mm² in the ADC-2 3 mg / kg group (T / C=14.08%, TGI=122.25%, p<0.001), the ADC-3 3 mg / kg group (T / C=33.43%, TGI=94.72%, p<0.001), the ADC-1 3 mg / kg group (T / C=46.15%, TGI=76.62%, p<0.001), and the DS1062a 3 mg / kg group (T / C=40.30%, TGI=84.96%, p<0.001), respectively. 3 The study demonstrated that all of the test drugs could significantly inhibit NCI-N87 tumor growth.

[0432] Example 3.2.3 Evaluation of in vivo efficacy of ADC-2 and ADC-3 for human pharyngeal squamous cell carcinoma (FaDu) In Example 3.2.1, BxPC-3 cells were exchanged for human pharyngeal squamous cell carcinoma FaDu, and 11 days after cell inoculation, the average tumor volume was approximately 123 mm².3 At this point, the animals were randomly divided into a vehicle control group, a DS1062a 3 mg / kg group, an ADC-1 3 mg / kg group, an ADC-2 3 mg / kg group, and an ADC-2 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-2 was evaluated using the same process as in Efficacy Example 3.2.1. The results are shown in Figure 13.1 and Table 18.

[0433] (Table 18) Inhibitory effect of ADC-2 on xenograft tumors in FaDu mice TIFF2026515759000195.tif81165

[0434] conclusion After 28 days of administration, the mean tumor volumes were 0, 4, 21, and 28 mm in the ADC-2 3 mg / kg group (T / C=0.00%, TGI=107.90%, p=0.003), the ADC-3 3 mg / kg group (T / C=0.25%, TGI=107.63%, p=0.003), the ADC-1 3 mg / kg group (T / C=1.23%, TGI=106.59%, p=0.003), and the DS1062a 3 mg / kg group (T / C=1.68%, TGI=106.11%, p=0.003), respectively. 3 This demonstrated that all of the test drugs could significantly inhibit FaDu tumor growth. Furthermore, at the end of the experiment, six mice in the 3 mg / kg ADC-2 group and three mice in the 3 mg / kg ADC-3 group showed complete tumor regression.

[0435] Efficacy Example 3.2.4 Evaluation of the in vivo efficacy of ADC-2 against MDA-MB-468 MDA-MB-468 tumor cells (ATCC, HTB-132) were maintained in vitro at 37°C under an atmosphere of 0% CO2 in L-15 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antifungal agent. Cells growing during the exponential growth phase were collected and counted for tumor inoculation. For in vivo antitumor efficacy testing, 10x10 cells were administered 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. After 24 days, the average tumor volume was 187 mm². 3 Upon reaching a certain stage, mice with tumors were randomly assigned and intravenously administered 0.5 mg / kg, 1.5 mg / kg, and 4.5 mg / kg of ADC-2, 4.5 mg / kg of Trodelvy, and 4.5 mg / kg of DS1062a. Tumor volume was measured twice weekly using calipers. T / C and TGI values ​​were calculated using tumor volume.

[0436] conclusion After 35 days of administration, the results were as follows: ADC-2 4.5 mg / kg group (T / C=0.00%, TGI=137.93%), DS1062a 4.5 mg / kg group (T / C=1.35%, TGI=136.06%), and Trodelvy 4.5 mg / kg group (T / C=78.73%). The results are shown in Figure 13.2 and the table below. ADC-2 showed significantly better efficacy than Trodelvy and slightly better efficacy than DS1062a.

[0437] Inhibitory effect of ADC-2 on xenograft tumors in MDA-MB-468 mice TIFF2026515759000196.tif81165

[0438] Efficacy Example 3.3 In vivo efficacy evaluation of ADC-5 and ADC-6 Efficacy 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, the average tumor volume was approximately 159 mm². 3 At this point, the animals were randomly divided into a vehicle control group, an ADC-5 3 mg / kg group, an ADC-6 3 mg / kg group, and a DS1062a 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-5 and ADC-6 was evaluated using the same process as in Efficacy Example 3.1.1. The results are shown in Figure 14 and Table 19.

[0439] (Table 19) Inhibitory effects of ADC-5 and ADC-6 on xenograft tumors in BxPC-3 mice TIFF2026515759000197.tif69165

[0440] conclusion After 42 days of administration, the mean tumor volume was 8 mm in the ADC-5 3 mg / kg group (T / C=0.75%, TGI=116.20%, p=0.003), the ADC-6 3 mg / kg group (T / C=2.48%, TGI=114.17%, p=0.003), and the DS1062a 3 mg / kg group (T / C=27.69%, TGI=84.60%, p=0.007), respectively. 3 , 27mm 3 , and 302mm 3 The study demonstrated that all of the test drugs could significantly inhibit tumor growth. The inhibitory effects of ADC-5 and ADC-6 were far superior to those of DS1062a.

[0441] Efficacy Example 3.3.2 Evaluation of in vivo efficacy of ADC-5 and ADC-6 against NCI-N87 gastric cancer cells In Example 3.3.1, BxPC-3 pancreatic cancer cells were exchanged for NCI-N87 gastric cancer cells. Six days after cell inoculation, the average tumor volume was approximately 196 mm². 3 At this point, the animals were randomly divided into a vehicle control group, an ADC-5 3 mg / kg group, an ADC-6 3 mg / kg group, and a DS1062a 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-5 and ADC-6 was evaluated using the same process as in Efficacy Example 3.3.1. The results are shown in Figure 15 and Table 20.

[0442] (Table 20) Inhibitory effects of ADC-5 and ADC-6 on xenograft tumors in NCI-N87 mice TIFF2026515759000198.tif81165

[0443] conclusion After 42 days of administration, the mean tumor volume was 133 mm in the ADC-5 3 mg / kg group (T / C=10.88%, TGI=106.06%, p<0.001), the ADC-6 3 mg / kg group (T / C=3.89%, TGI=114.36%, p<0.001), and the DS1062a 3 mg / kg group (T / C=35.67%, TGI=76.58%, p<0.001), respectively. 3 , 48mm 3 , and 437mm 3 The study demonstrated that all of the test drugs could significantly inhibit tumor growth. The inhibitory effects of ADC-5 and ADC-6 were far superior to those of DS1062a.

[0444] Efficacy Example 3.3.3 In vivo efficacy evaluation of ADC-5 and ADC-6 for human pharyngeal squamous cell carcinoma (FaDu) In Example 3.3.1, BxPC-3 pancreatic cancer cells were exchanged for human pharyngeal squamous cell carcinoma FaDu. Ten days after cell inoculation, the average tumor volume was approximately 119 mm². 3 At this point, the animals were randomly divided into a vehicle control group, an ADC-5 2 mg / kg group, an ADC-6 2 mg / kg group, and a DS1062a 2 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-5 and ADC-6 was evaluated using the same process as in Efficacy Example 3.3.1. The results are shown in Figure 16 and Table 21.

[0445] (Table 21) Inhibitory effects of ADC-5 and ADC-6 on xenograft tumors in FaDu mice TIFF2026515759000199.tif81165

[0446] conclusion After 31 days of administration, the mean tumor volume was 217 mm² in the ADC-5 2 mg / kg group (T / C=11.34%, TGI=94.56%, p=0.004), the ADC-6 2 mg / kg group (T / C=0.05%, TGI=106.60%, p=0.004), and the DS1062a 2 mg / kg group (T / C=15.47%, TGI=90.15%, p=0.005), respectively. 3 , 1mm 3 , and 296mm3 The study demonstrated that all of the test drugs could significantly inhibit tumor growth. The inhibitory effect of ADC-6 was far superior to that of DS1062a.

[0447] Example of effect 4: Serum stability of ADC-1 An appropriate amount of Trop 2 was covalently coupled to CNBr-activated agarose microspheres to form immobilized antigens. After blocking, a stable sample pre-incubated with plasma was added in a fixed proportion and incubated with shaking. ADC-1 and DS1062a (ADC drug) in the matrix were specifically captured by the immobilized antigen to form solid-phase antigen / antibody complexes, and unbound material was removed by washing. After incubation, the coupled glycans in the Fc region of the ADC drug antibody were excised using N-glycosidase, 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.

[0448] conclusion The results showed that the serum stability of ADC-1 was significantly better than that of DS1062a.

[0449] Example 5: In vivo efficacy evaluation of the combination of ADC2 and anti-mPD-1 in an MC38-hTROP2 syngeneic CDX model of colon cancer. Objective: To evaluate the in vivo antitumor efficacy of the combination of the ADC drug ADC2 and anti-mPD-1 in mice with a colon cancer Trop2 high CDX model.

[0450] I. MC38-hTROP2 cells (Biocytogen) in the exponential growth phase were collected and counted for tumor inoculation. 0.5 × 10⁶ cells were counted in 0.1 mL of PBS. 6 Individual cells were used and subcutaneously injected into the right flank of 6-8 week old SPF female C57BL / 6J mice.

[0451] II. Six days after vaccination, measure the diameter of the tumor with calipers, and use the formula V = 0.5a × b 2The tumor volume was calculated according to the following formula (where a is the longest diameter of the tumor and b is the shortest diameter). The average tumor volume was approximately 100-300 mm². 3 At that point, mice were randomized to one of the following groups: vehicle group, ADC2 3 mg / kg group, DS1062a 3 mg / kg group, anti-mPD-1 (Bio X cells, 825822J1) 1 mg / kg group, ADC2 + anti-mPD-1 combination group, and DS1062a + anti-mPD-1 combination group. Each group contained 6 mice. The day of the first administration was defined as day 0. Mice in the vehicle group were given the solvent for ADC2 and the solvent for anti-mPD-1 at the same frequency and via the same route of administration. Tumor volume of mice in each group was measured twice a week. The experiment was completed on day 28, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1 - (mean tumor volume of the treatment group on the end day - mean tumor volume of the treatment group on the first day) / (mean tumor volume of the vehicle group on the end day - mean tumor volume of the vehicle group on the first day)] × 100%.

[0452] III. Figure 18 shows the changes in tumor volume in tumor-bearing C57BL / 6J mice treated with (1) vehicle, (2) ADC2 3 mg / kg, (3) DS1062 3 mg / kg, (4) anti-mPD-1 1 mg / kg, (5) ADC2 3 mg / kg + anti-mPD-1 1 mg / kg, and (6) DS1062 3 mg / kg + anti-mPD-1 1 mg / kg. Table X shows the mean tumor volume on the final day (day 28) for the ADC2 3 mg / kg group, DS1062a 3 mg / kg group, anti-mPD-1 1 mg / kg group, ADC2 3 mg / kg + anti-mPD-1 1 mg / kg group, and DS1062a 3 mg / kg + anti-mPD-1 1 mg / kg group, all of which were 580 mm³. 3 , 1415mm 3 , 846mm 3 , 95mm 3 , and 934mm 3 The results showed that the TGIs were 68.6%, 12.8%, 50.8%, 101.0%, and 45.0%, respectively.

[0453] The results show that ADC2 3 mg / kg and anti-mPD-1 1 mg / kg can inhibit tumor cell proliferation as monotherapy. The combination of DS1062a and anti-mPD-1 inhibited tumor growth, and its effect was better than DS1062a monotherapy, but similar to anti-mPD-1 monotherapy. The combination of ADC2 and anti-mPD-1 showed excellent antitumor effects, resulting in 5 complete responses (5 / 6 CR), and had superior antitumor activity compared to ADC2 monotherapy (1 / 6 CR) or anti-PD-1 monotherapy.

[0454] (Table 22) Inhibition of tumor growth in the combination of ADC2 and anti-mPD-1 in a colon cancer syngeneic CDX model calculated by tumor volume. TIFF2026515759000200.tif88165

Claims

1. A combination pharmaceutical comprising a conjugate and an anti-PD-1 antibody, wherein the conjugate is of formula (III): It has a structure, During the ceremony, Q is hydrogen, -C ２ H ４ - (PEG) ｔ - (CO)NH ２ Or LKb-P, M is hydrogen or LKa-LKb-P, Each LKa operates independently. Selected from, opSu is, or a mixture thereof, Each LKb is independent of L ２ ―L １ —B, Each B either does not exist independently or is as follows: 1) a self-destructive spacer Sp1 and 2) a combination of one or a combination of two 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 -(CO)-, or a combination of two or more of said divalent groups, and preferably, B is -NH-CH ２ -U- or does not exist or -NH-CH ２ -U-(CR １ R ２ ) ｇ -(CO)-, and U either does not exist or is O, S, or NH, preferably O or S However, Q and M are not hydrogen at the same time. P is part B of equation (III) or L １ It is a payload that is joined to a part, Each L １ This 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 These can be independent, combined, or C ２～２０ It is an alkylene, where one or more -CH groups are present in the alkylene. ２ - Structure, - CR ３ R ４ -, -O-, -(CO)-, -S(=O) ２ -, -NR ５ - , C ４～１０ Cycloalkylene, C ４～１０ It may be replaced with heterocyclylene or phenylene, where the cycloalkylene, heterocyclylene, and phenylene are each independently unsubstituted or halogen, -C １～１０ Alkyl, -C １～１０ Haloalkyl, -C １～１０ Alkilen-NHR ８ and -C １～１０ Alkilen-OR ９ It is substituted with at least one substituent selected from the following: Ld2 and each Ld1 are independently bonded or -NH-C １～２０ Alkylene-(CO)-,-NH-(PEG) ｉ Selected from -(CO)-, or a natural amino acid or natural oligomeric amino acid having a degree of polymerization of 2 to 10, wherein the natural amino acid or natural oligomeric amino acid is independently unsubstituted or has -(PEG) in its side chain. ｊ -R １１ It has been replaced with, - (PEG) ｔ -, - (PEG) ｉ - and - (PEG) ｊ Each of the hyphens represents a sequence of hyphens (O-C) of the indicated number. ２ H ４ ) - Structural unit or continuous - (C ２ H ４ -O)- Includes a structural unit and an optional additional C at one end. １～１０ A PEG fragment containing alkylene R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ These are, independently, hydrogen, halogen, and -C. １～１０ Alkyl, -C １～１０ Haloalkyl, C ４～１０ Selected from cycloalkylenes, or R １ and R ２ They, together with the carbon atoms to which they are bonded, form a 3- to 6-membered cycloalkyl group, or R ３ and R ４ Together with the carbon atoms to which they are bonded, they form a 3- to 6-membered cycloalkyl group. R １１ is C １～１０ It is alkyl, m is any integer between 1 and 3. n is any integer between 2 and 20. d is either 0 or any integer between 1 and 6. Each i is an independent integer between 0 and 100, preferably between 0 and 20, and preferably each i is an independent integer between 0 and 12, more preferably between 0 and 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. A is an anti-TROP2 antibody or its antigen-binding fragment, preferably G of formula (III). ｎ It has been modified to bond with a part, and G is glycine. z is an integer between 1 and 20. The aforementioned combination drug.

2. The aforementioned conjugate is given by the following equation (III-a) or equation (III-b): The combination pharmaceutical according to claim 1, having the structure described above.

3. The aforementioned conjugate is as follows: It has a structure, Preferably, z is 1 to 4, preferably 2. Each i, i1, i2, i3, i4 is independently an integer between 0 and 100, preferably between 0 and 20, and preferably each i, i1, i2, i3, i4 is independently an integer between 0 and 12, more preferably between 0 and 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. Preferably, n is 3, L ２ is, -(CH ２ ) ｐ - (CH ２ ) ２ (CO)- or -(C ２ H ４ -O) ｐ - (CH ２ ) ２ (CO)-, p is 2-4, L １ is Gly-Gly-Phe-Gly, and B is -NH-CH ２ -U-, or nonexistent, or -NH-CH ２ -U-(CR １ R ２ ) ｇ It is -(CO)-, U does not exist, or U is O, and g is 1. 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 any integer between 1 and 3, especially 1 or 2. The combination pharmaceutical according to claim 1 or 2.

4. The payload is a cytotoxin or a fragment thereof, and is the B portion or L portion of the compound of formula (III) as defined in claim 1. １ They may be derivatized to bind to the portion, Preferably, the cytotoxin is Taxanes, mytansinoids, auristatin, eposylone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinblastines, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunin, vinglycinate, anhydrovinblastine, drastatin 10 and its analogs, halichondrin B, eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermold, laurimalide, camptothecin and its derivatives, mitoxantrone, mitoggua Zon, Nitrogen Mustard, Nitrosourea, Aziridine, Benzodopa, Carbocon, Metsuredepa, Uredepa, Dynemycin, Esperamicin, Neocartinostatin, Acrasinomycin, Actinomycin, Anthramycin, Bleomycin, Actinomycin C, Carabicin, Carminomycin, Cardinophilin, Carminomycin, Actinomycin D, Daunorubicin, Detrubicin, Adriamycin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin, Nogaramycin, Olibomycin, Peplomycin, Porphyromycin, Puromycin, Iron Adriamycin (ferric adriamycin), Rhodolubicin, Ruhochromomycin, Streptozocin, Dinostatin, Zolubicin, Trichothecene, T-2 Toxin, Veracurin AA) Selected from the group consisting of basilocuporin A, anguidin, ubenimex, azaserin, 6-diazo-5-oxo-L-norleucine, dimethyl folate, methotrexate, pteropterin, trimethrexate, edatrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine, carsterone, dromostanolone propionate, epithiostanol, mepithiostan, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitors, and proteasome inhibitors, and / or Selected from and / or from vinblastine, colchicine, taxane, auristatin, mytansinoid, calicheamicin, doxorubicin, duocarmycin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, drastancin, pyrrolobenzodiazepine, exatecan, and their derivatives, and / or Auristatin, particularly selected from MMAE, MMAF, or MMAD, and / or Selected from exatecan and its derivatives, for example, DX8951f, A combination pharmaceutical product according to any one of claims 1 to 3.

5. The aforementioned payload is given by formula (i): It has a structure, During the ceremony, a* is either 0 or 1, The carbon atoms marked with p1* and p2* are chiral centers, and these chiral centers are in an S configuration, an R configuration, or a racemic mixture. L １＊ is an unsubstituted C １～６ Alkylene, or halogen, -OH, and -NH ２ C substituted with one substituent selected from １～６ Selected from alkylenes, M* is -CH ２ -, -NH-, or -O- L ２＊ C １～３ It is alkylene, R １＊ and R ２＊ each independently is hydrogen, C １～６ alkyl, halogen, and C １～６ selected from alkoxy, A combination pharmaceutical product according to any one of claims 1 to 4.

6. L １＊ is C １～６ a linear alkylene, C １～６ a branched alkylene, C ３～６ a cyclic alkylene and C ３～４ a cyclic alkyl-C １～２ selected from a linear alkylene group, where the alkylene and cyclic alkylene are each independently unsubstituted or substituted with one substituent selected from halogen, -OH and -NH ２ and preferably, L １＊ is selected from C １～４ an alkylene, where the alkylene is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH ２ and more preferably, L １＊ is -CH ２ -, -C ２ H ４ - Selected from these, each independently is either unsubstituted or contains halogen, -OH, and -NH ２ It is substituted with at least one substituent selected from, most preferably L １＊ However, -CH ２ - The combination pharmaceutical according to claim 5, wherein selected from, and in the formula, "#" indicates the position of bond to the carbonyl group.

7. A combination pharmaceutical according to any one of claims 5 to 6, wherein a* is 0.

8. R １＊ However, C １～６ Selected from alkyl and halogen, preferably R １＊ The combination pharmaceutical according to any one of claims 5 to 7, wherein the compound is methyl or Cl.

9. R ２＊ However, C １～６ Selected from alkyl and halogen, preferably R ２＊ A combination pharmaceutical according to any one of claims 5 to 8, wherein F is

10. The aforementioned payload, Selected from, especially, Selected from, A combination pharmaceutical product according to any one of claims 1 to 3.

11. The aforementioned conjugate, Selected from, Each g is an integer from 1 to 6, preferably from 1 to 3, and more preferably 1. Each R １ and R ２ However, independently, hydrogen, halogen, -C １～１０ Alkyl, -C １～１０ Haloalkyl, C ４～１０ Selected from cycloalkylenes, or R １ and R ２ However, together with the carbon atoms to which they are bonded, they form a 3- to 6-membered cycloalkyl group, preferably R １ and R ２ That is hydrogen, Each t is an integer from 1 to 100, preferably from 1 to 20, preferably from 1 to 12, more preferably from 8 to 12, particularly 8 or 12. m is any integer between 1 and 3, especially 1 or 2. z is an integer from 1 to 20, particularly 2 or 4, more preferably 2. A combination pharmaceutical product according to any one of claims 1 to 3.

12. The antibody or its antigen-binding fragment has a heavy chain variable region (V Ｈ ) and light chain variable region (V Ｌ ) including, The aforementioned V Ｈ but, (i) X １ X ２ GMX ３ (Sequence ID 1) contains the amino acid sequence, where X １ is N, T, or A, and X ２ is Y or A, X ３ HCDR1 is N or Q, (ii) WINTX ４ X ５ GX ６ PX ７ YX ８ X ９ The amino acid sequence of DFKG (SEQ ID NO: 2) is included, where X ４ is Y, H, or D, and X ５ is T or S, X ６ However, it is either E or V, and X ７ is T or K, X ８ is T or A, X ９ HCDR2 is D or E, (iii)X １０ The amino acid sequence GFGSSYWYFDV (SEQ ID NO: 3) is included, where X １０ HCDR3 is G or S Includes, and / or, The aforementioned V Ｌ but, (i) LCDR1 containing the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSSTAVA (SEQ ID NO: 14), (ii) LCDR2 containing the amino acid sequence of SASYRYT (SEQ ID NO: 15), (iii)LCDR3 containing the amino acid sequence of QQHYITPLT (SEQ ID NO: 16) including, The combination pharmaceutical product according to claim 1.

13. The aforementioned V Ｈ but, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, (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 Includes, and / or, The aforementioned V Ｌ but, (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 Does it include, or, The aforementioned V Ｈ but, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 5, (ii) HCDR2 containing the amino acid sequence of SEQ ID NO: 9, and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 12 Includes, and / or, The aforementioned V Ｌ but, (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 14, (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 Does it include, or, The aforementioned V Ｈ but, (i) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, (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 Includes, and / or, The aforementioned V Ｌ but, (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 Does it include, or, The aforementioned V Ｈ but, (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 Includes, and / or, The aforementioned V Ｌ but, (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 Does it include, or, The aforementioned V Ｈ but, (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 Includes, and / or, The aforementioned V Ｌ but, (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 including, The combination pharmaceutical according to claim 12.

14. The aforementioned V Ｈ However, it contains an amino acid sequence that has at least approximately 90% sequence identity with the amino acid sequences of SEQ ID NOs. 21-25, and / or The aforementioned V Ｌ However, it includes an amino acid sequence that has at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO:

27. The combination pharmaceutical product according to claim 12 or 13.

15. The antibody or antigen-binding fragment is A heavy chain constant domain (CH) containing an amino acid sequence having at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 28, and / or Light chain constant domain containing an amino acid sequence having at least approximately 90% sequence identity with the amino acid sequence of SEQ ID NO: 29 A combination pharmaceutical product according to any one of claims 12 to 14, including the combination pharmaceutical product described in any one of claims 12 to 14.

16. The antibody or antigen-binding fragment has an equilibrium dissociation constant (K) of approximately 0.5 nM to approximately 20 nM. Ｄ The combination pharmaceutical according to any one of claims 12 to 15, wherein it binds to TROP2.

17. The combination pharmaceutical according to claim 12, wherein the antibody or antigen-binding fragment comprises a heavy chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs. 30 to 33, and / or a light chain containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NOs. 34 or 35.

18. The combination pharmaceutical according to claim 17, wherein the antibody or antigen-binding fragment includes a C-terminal modification of the heavy chain and / or a C-terminal modification of the light chain such that the recognition sequences of the antibody, Sp, and ligated donor substrate are sequentially bound, Sp is a spacer sequence selected from GA, GGGGS, GGGGSGGGGGS and GGGGSGGGGGGGS, and the recognition sequence of the ligated donor substrate is LPXTGJ, where X may be any single amino acid, natural or non-natural, and J may be absent or an amino acid fragment containing 1 to 10 amino acids.

19. The modified antibody or its antigen-binding fragment contains the heavy chain of SEQ ID NOs. 30-33 and / or the light chain of SEQ ID NOs. 40 or 41. or The modified antibody or its antigen-binding fragment contains the heavy chain of SEQ ID NOs. 36-39 and / or the light chain of SEQ ID NOs. 34 or 35. The combination pharmaceutical product according to claim 18.

20. The combination pharmaceutical according to claim 1, wherein the conjugate has a drug-to-antibody ratio (DAR) of an integer or non-integer between 1 and 19.

21. The anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody, and / or The anti-PD-1 antibody binds to human FFFR3 and / or monkey FFFR3 and / or mouse FFFR3, or the anti-PD-1 antibody binds to human FFFR3 and monkey FFFR3 but not to mouse FFFR3. A combination pharmaceutical product according to any one of claims 1 to 20.

22. The combination pharmaceutical according to claim 21, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, tripalimab, tislerizumab, cintilimab, and camrelizumab.

23. A combination pharmaceutical according to any one of claims 1 to 22, further comprising, optionally, a pharmaceutically acceptable carrier.

24. A kit comprising a combination pharmaceutical according to any one of claims 1 to 23.

25. A first packaging unit comprising a conjugate as defined in any one of claims 1 to 23, A second packaging unit comprising an anti-PD-1 antibody as defined in any one of claims 1 to 23, Instructions for administering the conjugate and anti-PD-1 antibody to the target of optional administration are provided. The kit according to claim 24, including the following:

26. Use of a combination pharmaceutical according to any one of claims 1 to 23 or a kit according to claim 24 or 25 in the manufacture of a drug for treating a disease that is a TROP2-related tumor.

27. The use according to claim 26, wherein the TROP2-related tumor includes tumors that overexpress TROP2 or tumors having a TROP2 gene mutation.

28. The use according to claim 27, wherein the tumor includes breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

29. A method for treating a subject suffering from a disease or for reducing the likelihood of disease progression, comprising administering a combination pharmaceutical according to any one of claims 1 to 23, or a kit according to claim 24 or 25, wherein the disease is a tumor.

30. A method for treating a subject suffering from cancer or for reducing the likelihood of cancer progression, comprising administering an effective amount of a conjugate according to any one of claims 1 to 20 to the subject, and administering an effective amount of an anti-PD-1 antibody to the subject.

31. The method according to claim 30, wherein the cancer overexpresses TROP2 or has a TROP2 gene mutation.

32. The method according to claim 30 or 31, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

33. The anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody, and / or The anti-PD-1 antibody binds to human FFFR3 and / or monkey FFFR3 and / or mouse FFFR3, or the anti-PD-1 antibody binds to human FFFR3 and monkey FFFR3 but not to mouse FFFR3. The method according to any one of claims 30 to 32.

34. The method according to any one of claims 30 to 32, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, tripalimab, tislerizumab, cintilimab, and camrelizumab.

35. The aforementioned conjugate, The method according to any one of claims 30 to 34, wherein the conjugate is ADC-2.

36. The method according to any one of claims 30 to 35, wherein the conjugate and the anti-PD-1 antibody are administered simultaneously as part of the same pharmaceutical preparation.

37. The method according to any one of claims 30 to 35, wherein the conjugate and the anti-PD-1 antibody are administered simultaneously as part of different pharmaceutical formulations.

38. The method according to any one of claims 30 to 35, wherein the conjugate and the anti-PD-1 antibody are administered at different time points.

39. Use of an effective amount of the conjugate according to any one of claims 1 to 20 for the manufacture of a drug for the treatment of subjects with cancer, used in combination with an effective amount of anti-PD-1 antibody.

40. The use according to claim 39, wherein the cancer overexpresses TROP2 or has a TROP2 gene mutation.

41. The use according to claim 39 or 40, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

42. The anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody, and / or The anti-PD-1 antibody binds to human FFFR3 and / or monkey FFFR3 and / or mouse FFFR3, or the anti-PD-1 antibody binds to human FFFR3 and monkey FFFR3 but not to mouse FFFR3. The use according to any one of claims 39 to 41.

43. The use according to any one of claims 39 to 41, wherein the anti-PD-1 antibody is selected from pembrolizumab, nivolumab, tripalimab, tislerizumab, cintilimab, and camrelizumab.

44. The aforementioned conjugate, The use according to any one of claims 39 to 43, wherein the conjugate is ADC-2.

45. The use according to any one of claims 39 to 44, wherein the conjugate and the anti-PD-1 antibody are intended to be administered simultaneously as part of the same pharmaceutical preparation.

46. The use according to any one of claims 39 to 44, wherein the conjugate and the anti-PD-1 antibody are intended to be administered simultaneously as part of different pharmaceutical formulations.

47. The method according to claims 39 to 44, wherein the conjugate and the anti-PD-1 antibody are to be administered at different time points.

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