Combinations of antibody-drug conjugates and anti-PD-1 antibodies and uses thereof

Abstract

The present invention relates to a combination of an antibody-drug conjugate and an anti-PD-1 antibody and uses thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biomedicine, in particular to the combination of antibody-drug conjugate and anti-PD-1 antibody and uses thereof. BACKGROUND

[0002] TROP2 is a transmembrane protein and has been found to be overexpressed in several cancer types, including endometrial cancer, prostate cancer, pancreatic cancer, colon cancer, gastric cancer, oral cancer, glioma, making TROP2 a natural candidate for the development of targeted therapies. TROP2 acts as a modulator in cell self-renewal, proliferation, and transformation. Experiments have shown that TROP2 can promote tumor growth and knocking out the TROP2 gene interferes with tumor cell proliferation. The expression of TROP2 in tissues is limited, thus reducing the toxicity of treatment, which is also an advantage of targeting TROP2 therapy. Multiple TROP2-targeted ADCs have been proposed, such as DS-1062, TRODELVY, BAT8003. These therapeutic agents significantly improve the survival rate of TROP2-positive cancer patients.

[0003] DS-1062 is an antibody-conjugated drug developed by Daiichi Sankyo using its proprietary DXd ADC technology. DS-1062 consists of a monoclonal antibody targeting Trop2 protein, which is linked to DXd. Data including more NSCLC patients show that DS-1062 exhibits good dose-dependent anticancer activity. With increasing dose, more NSCLC patients have their tumors shrink. TRODELVY is the first FDA-approved ADC specifically for recurrent or refractory metastatic TNBC, and also the first FDA-approved anti-TROP2 ADC. TRODELVY consists of an antibody targeting TROP2, which is linked to SN-38 (the active metabolite of the chemotherapy drug irinotecan).

[0004] However, there is still a huge demand for new ADC drugs targeting TROP2, and ADCs with high safety are also one of the directions of new drug development. SUMMARY

[0005] In a first aspect, there is provided a compound of formula (I):

[0006]

[0007] wherein,

[0008] W is hydrogen, LKb, or -C2H4-(PEG) t -(CO)NH2;

[0009] Y is hydrogen or LKa-LKb;

[0010] provided that W and Y are not simultaneously hydrogen;

[0011] each LKa is independently selected from opSu is or mixtures thereof;

[0012] each LKb is independently L 2 -L 1 -B;

[0013] each B is independently a terminal group R 10 or a combination of 1), 2) and 3) below: 1) a self-cleaving spacer Sp1; 2) a bond or one or a combination of two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) a terminal group R 10 ;

[0014] R 10 is hydrogen or a group that can be left upon reaction with a group in the support;

[0015] L 1 is a cleavable sequence 1 comprising an amino acid sequence that can be cleaved by an enzyme, and the cleavable sequence 1 comprises 1-10 amino acids;

[0016] L 2 is a bond; or C 2-20 alkylene, wherein one or more -CH2- structure in the alkylene is optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, -N⊕R 6 R 7 -, C 4-10 cycloalkylene, C 4-10 heterocyclylene, phenylene; wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from the group consisting of halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R 9 ;

[0017] Ld2 and each Ld1 are independently a bond; or selected from -NH-C1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-, or is a natural amino acid or an oligomeric natural amino acid having a degree of polymerization of 2-10, which natural amino acid or oligomeric natural amino acid is independently unsubstituted or substituted on the side chain by -(PEG) j -R 11 substituted;

[0018] -(PEG) t -, -(PEG) i -, and -(PEG) j - are each a PEG fragment comprising the indicated number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units with an optional additional C 1-10 alkylene;

[0019] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from the group consisting of hydrogen, halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, C 4-10 cycloalkylene; or

[0020] R 1 and R 2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group; or

[0021] R 3 and R 4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group;

[0022] R 11 is C 1-10 alkyl;

[0023] m is any integer from 1 to 3;

[0024] n is any integer from 2 to 20;

[0025] d is 0, or is any integer from 1 to 6; each i is independently an integer from 0 to 100, preferably from 0 to 20; preferably, each i is independently an integer from 0 to 12; more preferably from 0 to 8; in particular 4;

[0026] each j is independently an integer from 1 to 100, preferably 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably 8 to 12; in particular 8 or 12;

[0027] each t is independently an integer from 1 to 100, preferably 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably 8 to 12; in particular 8 or 12.

[0028] In a second aspect, there is provided a compound having the structure of formula (II)

[0029]

[0030] wherein

[0031] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2or LKa-LKb―P;

[0032] M is hydrogen or LKa-LKb―P;

[0033] with the proviso that Q and M are not simultaneously hydrogen;

[0034] P is a payload, said payload being linked to the B moiety or L 1 of the compound of formula (I);

[0035] n, d, Ld1, Ld2, t, LKaand LKbare as defined in formula (I);

[0036] Preferably, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or a combination of 1) a self-cleaving spacer Sp1; and 2) a bond or one or a combination of two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0037] Preferably, Sp1is selected from the group consisting of PABC, acetal, heteroacetal and combinations thereof; more preferably, Sp1is an acetal, a heteroacetal or PABC; further preferably, the heteroacetal is selected from the group consisting of N,O-heteroacetals; more preferably, Sp1is -O-CH2-U- or -NH-CH2-U-; wherein the -O- or -NH- is linked to the cleavable sequence 1 and U is absent, or is O, S or NH, preferably O or S.

[0038] In a third aspect, an anti-TROP2 antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein

[0039] the V H comprises:

[0040] (i) a HCDR1 comprising the amino acid sequence of X1AGMN (SEQ ID NO: 45), wherein X1is N or A;

[0041] (ii) a HCDR2 comprising the amino acid sequence of WINTDSGEPTYTDDFKG (SEQ ID NO: 10) or WINTYTGEPTYTDDFKG (SEQ ID NO: 8); and

[0042] (iii) a HCDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 11); and / or

[0043] the V L comprises:

[0044] (i) a LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0045] (ii) a LCDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 15); and

[0046] (iii) a LCDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 16).

[0047] In a fourth aspect, a cytotoxin having the structure of formula (i) is provided:

[0048]

[0049] wherein,

[0050] a* is 0 or 1 ;

[0051] the carbon atoms labeled pi*and p2*are each asymmetric centers and the asymmetric centers are either the S configuration, the R configuration, or racemic;

[0052] L 1* is selected from C 1-6 alkylene, the C 1-6alkylene is unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2;

[0053] M* is -CH2-, -NH- or -O-;

[0054] L 2* is C 1-3 alkylene;

[0055] R 1* and R 2* are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0056] In a fifth aspect, a pharmaceutical combination comprising a conjugate and an anti-PD-1 antibody is provided, wherein the conjugate has the structure of Formula (III):

[0057]

[0058] wherein,

[0059] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb―P;

[0060] M is hydrogen or LKa-LKb―P;

[0061] each LKa is independently selected from

[0062] opSu is or mixtures thereof;

[0063] each LKb is independently L 2 ―L 1 ―B;

[0064] m, n, d, Ld1, Ld2, t, LKa and LKb are as defined in Formula (I);

[0065] each B is independently absent, or a combination of 1) a self-cleaving spacer Sp1; and 2) a bond or one or a combination of two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclyl and -(CO)-; preferably, B is -NH-CH2-U-, absent, -NH-CH2-U-(CR 1 R 2 ) g-(CO)- or NH-CH2-U-(CH2) g -(CO)-;

[0066] with the proviso that Q and M are not simultaneously hydrogen;

[0067] P is a support, said support being attached to the B moiety or L 1 moiety of the compound of formula (I);

[0068] each L 1 is independently a cleavable sequence 1 comprising an amino acid sequence that is cleavable by an enzyme, and said cleavable sequence 1 comprises 1-10 amino acids;

[0069] each L 2 is independently a bond; or C 2-20 alkylene, wherein one or more -CH2- structures in said alkylene are optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, -N⊕R 6 R 7 -, C 4-10 cycloalkylene, C 4-10 heterocyclylene, phenylene; wherein said cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from the group consisting of halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R 9 ;

[0070] -(PEG) t -, -(PEG) i - and -(PEG) j each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- or -(C2H4-O)- structural units, with optional additional C 1-10 alkylene at one end;

[0071] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 each independently is selected from the group consisting of hydrogen, halogen, -C 1-10alkyl, -C 1-10 haloalkyl, C 4-10 cycloalkylene; or

[0072] R 1 and R 2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group; or

[0073] R 3 and R 4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group;

[0074] 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; in particular 4;

[0075] 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; in particular 8 or 12;

[0076] 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; in particular 8 or 12;

[0077] each g is independently an integer from 1 to 10, e.g. 1, 2, 3, 4, 5, 6, 7;

[0078] A is an anti-TROP2 antibody or antigen binding fragment thereof attached to G n of the compound of formula (I); G is glycine;

[0079] z is an integer from 1 to 20.

[0080] In some embodiments, the pharmaceutical combination further comprises at least one pharmaceutically acceptable carrier.

[0081] In another aspect, a kit comprising the pharmaceutical combination is provided.

[0082] In another aspect, use of the pharmaceutical combination or the kit in the manufacture of a medicament for the prevention or treatment of a disease; wherein the disease is a tumor is provided.

[0083] In another aspect, a method of treating a subject afflicted with a disease or preventing progression of a disease is provided, the method comprising administering the pharmaceutical combination or the kit; and the disease is a tumor.

[0084] In one embodiment, the tumor is a TROP2-related tumor.

[0085] In some embodiments, the disease is a tumor. In some embodiments, the disease comprises a TROP2 positive tumor. In some embodiments, the disease comprises a tumor that overexpresses TROP2 or a tumor that has a mutation in the TROP2 gene. In some embodiments, the disease is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, synovial sarcoma, hemangiosarcoma, hemangioendothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma. In one embodiment, the TROP2 associated tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial cancer.

[0086] In another aspect, there is provided a method of treating a subject having a cancer or reducing the likelihood of progression of a cancer, 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.

[0087] In another aspect, there is provided the use of an effective amount of the conjugate in combination with an effective amount of an anti-PD-1 antibody in the manufacture of a medicament for treating a subject having a cancer. BRIEF DESCRIPTION OF DRAWINGS

[0088] Figure 1.1 Internalization of antibody on MBA-MD-468 is shown, Figure 1.2 Binding activity on Trop2 ECD is shown, Figure 1.3 Internalization on NCI-N87 is shown.

[0089] Figure 2 Effect of ADC-7 and DS1062a on FaDu cell viability is shown.

[0090] Figure 3 Effect of ADC-1 and DS1062a on human pancreatic cancer cell BxPC-3 viability is shown.

[0091] Figure 4 Effect of ADC-1 and DS1062a on human breast cancer cell MDA-MB-468 viability is shown.

[0092] Figure 5 The effect of ADC-7 and DS1062a on the viability of gastric cancer cells NCI-N87 is shown.

[0093] Figure 6 The effect of ADC-2, ADC-3 and ADC-1 on the viability of pharyngeal squamous cell carcinoma FaDu is shown.

[0094] Figure 7.1 The effect of ADC-2, ADC-3 and ADC-1 on the proliferation of human pancreatic cancer cells BxPC-3 is shown, Figure 7.2 The effect of ADC-2 on the proliferation of BxPC-3 is shown, Figure 7.3 The effect of ADC-2 on the proliferation of FaDu is shown, Figure 7.4 The effect of ADC-2 on the proliferation of NCI-N87 is shown.

[0095] Figure 8 The inhibitory effect of ADC-1 on BxPC-3 mouse xenograft tumors is shown.

[0096] Figure 9 The inhibitory effect of ADC-1 on NCI-N87 mouse xenograft tumors is shown.

[0097] Figure 10 The inhibitory effect of ADC-1 on BR-05-0028 mouse xenograft tumors is shown.

[0098] Figure 11 The inhibitory effect of ADC-2 and ADC-3 on BxPC-3 mouse xenograft tumors is shown.

[0099] Figure 12 The inhibitory effect of ADC-2, ADC-3 and ADC-1 on NCI-N87 mouse xenograft tumors is shown.

[0100] Figure 13.1 The inhibitory effect of ADC-2, ADC-3 and ADC-1 on FaDu mouse xenograft tumors is shown; Figure 13.2 The inhibitory effect of ADC-2 on MDA-MB-468 mouse xenograft tumors is shown.

[0101] Figure 14 The inhibitory effect of ADC-5 and ADC-6 on BxPC-3 mouse xenograft tumors is shown.

[0102] Figure 15 The inhibitory effect of ADC-5 and ADC-6 on NCI-N87 mouse xenograft tumors is shown.

[0103] Figure 16Inhibition of FaDu mouse xenograft tumors by ADC-5 and ADC-6 is shown.

[0104] Figure 17 Serum stability results for ADC-1 are shown.

[0105] Figure 18 Combination of ADC2 with anti-mPD-1 in MC38-hTROP2 colon cancer syngeneic CDX model is shown. DETAILED DESCRIPTION

[0106] The specific embodiments provided below are illustrative of the technical content of this document. Other advantages and effects will be readily appreciated by those skilled in the art from the disclosure contained herein. The technical content herein can also be embodied in other different specific embodiments or applied to other different specific embodiments. Various modifications and changes can be made thereto without departing from the essence of the technical content herein.

[0107] Definitions

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this document belongs. The techniques used herein are those that are generally known in the art, including variations and equivalents thereof, which are apparent to those skilled in the art. Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to better illustrate the present document. When a name, trademark, and the like is present in this document, it is intended to convey the corresponding product or active ingredient thereof. All patents, published patent applications, and publications recited herein are incorporated herein by reference.

[0109] When a range, preferably a range, or a preferred upper or lower limit is listed, it is understood that it is equivalent to any range formed by combining any upper or preferred value with any lower or preferred value, whether or not the range is explicitly listed. Unless otherwise stated, numerical ranges recited herein are intended to include the end points of the range and all integers and fractions (decimals) within the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions (such as j, k, and g) should also be understood in a similar manner.

[0110] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The expressions "one or more" or "at least one" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0111] The terms "about" and "approximately" when used in connection with a numerical variable generally mean that the value of the variable and all values of the variable that are within an experimental error (e.g., within a 95% confidence interval of the mean) or within ± 10% of the indicated value, or a wider range.

[0112] The term "stoichiometric ratio" refers to the proportioning of various substances in a particular weight ratio. For example, herein, active ingredients are mixed with fillers, binders, and lubricants in the specified weight ratios.

[0113] The terms "optional" or "optionally" mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the stated event or circumstance occurs and instances where it does not.

[0114] The expressions "comprising", "including", "containing", and "having" are open-ended, and do not exclude additional, unrecited elements, steps, or ingredients. The expression "consisting of" excludes any element, step, or ingredient not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or ingredients, and to other elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It is understood that the expression "comprising" encompasses the expressions "consisting of" and "consisting essentially of."

[0115] The term "antibody", as used herein, is used in the broadest sense of the term and specifically includes intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. The antibodies can be of any subclass (such as IgG, IgE, IgM, IgD, and IgA) or class, and can be derived from any suitable species. In some embodiments, the antibodies are human- or murine-derived. The antibodies can also be fully human, humanized, or chimeric antibodies produced by recombinant methods.

[0116] Monoclonal antibodies are used herein to refer to antibodies obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The word "monoclonal" refers to the character of the antibodies as produced from a substantially homogeneous population of antibodies, and not to the method by which the antibodies are produced.

[0117] A complete antibody or full-length antibody essentially comprises an antigen binding variable region as well as a light chain constant region (CL) and a heavy chain constant region (CH), which can include CH1, CH2, CH3, and / or CH4, depending on the antibody subtype. The antigen binding variable region (also referred to as variable region fragment, Fv fragment) generally comprises a light chain variable region (VL) and a heavy chain variable region (VH). The constant region can be a constant region having a native sequence (such as a constant region having a human native sequence), or can be a constant region having an amino acid sequence variant of the native sequence. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0118] An antibody fragment can comprise a portion of a complete antibody, preferably an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementarity determining regions (CDRs). A Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin, or a fragment having the same structure produced, for example, by recombinant expression. A Fab fragment comprises a light chain (comprising VL and CL) and another chain, wherein the other chain comprises a variable domain of a heavy chain (VH) and a constant region domain of a heavy chain (CH1). An F(ab')2 fragment is an antibody fragment obtained by pepsin digestion of an immunoglobulin at pH 4.0 to 4.5, or a fragment having the same structure produced, for example, by recombinant expression. An F(ab')2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises a small number of additional amino acids, including cysteine forming a disulfide bond connecting the two fragments. A Fab' fragment (one heavy chain and one light chain) is a fragment comprising one half of an F(ab')2 fragment. An antibody fragment can comprise multiple chains joined together, for example, via disulfide bonds and / or via a peptide linker. Examples of antibody fragments also include single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments. An antibody fragment generally comprises at least or about 50 amino acids, and generally comprises at least or about 200 amino acids. An antigen binding fragment can include any such antibody fragment that, when inserted into an antibody framework region (e.g., by substitution of the corresponding region), is capable of producing an antibody that immunospecifically binds an antigen.

[0119] Antibodies according to the present disclosure can be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimetics) can be expressed by suitable culture systems (e.g., E. coli or mammalian cells). Engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0120] Cytotoxin refers to a substance that inhibits or prevents the expression of an activity, a cellular function, and / or causes destruction of a cell. Cytotoxins used in ADCs are currently more toxic than chemotherapeutic drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, regulation of apoptosis. Drugs targeting the microtubule cytoskeleton can be, for example, microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinca alkaloids, colchicines, and dolastatins. DNA-targeting drugs can be, for example, drugs that directly damage DNA structure or topoisomerase inhibitors. Examples of drugs that directly damage DNA structure include, but are not limited to, DNA double-strand breakers, DNA alkylators, DNA intercalators. DNA double-strand breakers can be, for example, enediyne antibiotics, including but not limited to dynemicin, esperamicin, neocarzinostatin, uncialamycin, and the like. DNA alkylators can be, for example, DNA bisalkylators (i.e., DNA crosslinkers) or DNA monoalkylators. Examples of DNA alkylators include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, indolinobenzodiazepine (IGN) dimers, duocarmycin-like compounds, and the like. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and derivatives thereof (such as DX8951f, DXd-(1), and DXd-(2), the structures of which are shown below), camptothecin, and anthracyclines. RNA-targeting drugs can be, for example, drugs that inhibit splicing, examples of which include, but are not limited to, pladienolide. Drugs targeting kinesin-mediated protein transport can be, for example, mitotic kinesin inhibitors, including but not limited to kinesin spindle protein (KSP) inhibitors.

[0121] A spacer is a structure located between different structural modules and capable of spatially separating these modules. The definition of a spacer is not limited by whether it has a 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, where non-amino acid structures can be, but are not limited to, amino acid derivatives or analogs. A “spacer sequence” refers to an amino acid sequence used as a spacer, and examples include, but are not limited to, single amino acids, sequences containing multiple amino acids (e.g., 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-cleaving spacer is a covalent component that causes the two chemical bonds in a precursor to cleave sequentially upon activation of the protective moiety: upon stimulation, the protective moiety (such as a cleavable sequence) is removed, resulting in a cascade of decomposition reactions that lead to the sequential release of small molecules. Examples of self-cleaving spacers include, but are not limited to, PABC (p-benzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0122] The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, connected to the rest of the molecule by single bonds. Alkyl groups can contain 1 to 20 carbon atoms, involving C1-C2. 20 Alkyl groups, such as C1-C4 alkyl groups, C1-C3 alkyl groups, C1-C2 alkyl, C3 alkyl, C4 alkyl, 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 is a group obtained from a corresponding monovalent radical by removing a hydrogen atom from a carbon atom that has free valence electrons. A divalent radical has two connection sites that attach to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a straight-chain or branched saturated divalent hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H6-). 10 -), Hexyl (-C6H) 12), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, and the like.

[0123] As used herein, when a group is combined with another group, the attachment of those groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such combination can be attached to the rest of the molecule via any suitable atom in the structure, preferably via the indicated chemical bond. For example, when two or more divalent groups selected from -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and -(CO)- are combined together to form a combination, the two or more divalent groups can form a linear attachment to each other, such as -CR 1 R 2 -C 1-10 alkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-C 1-10 alkylene-(CO)-, -CR 1 R 2 -CR 1' R 2' -(CO)-, -CR 1 R 2 -CR 1' R 2' -CR 1” R 2” -(CO)-, and the like. The resulting divalent structure can be further attached to the rest of the molecule.

[0124] As used herein, the expressions "antibody-conjugated drug" and "antibody-drug conjugate" have the same meaning.

[0125] Compound of formula (I)

[0126] In one aspect, a compound of Formula (I) is provided:

[0127]

[0128] wherein,

[0129] W is hydrogen, LKb, or -C2H4-(PEG) t -(CO)NH2;

[0130] Y is hydrogen or LKa-LKb;

[0131] with the proviso that W and Y are not simultaneously hydrogen;

[0132] each LKa is independently selected from

[0133] opSu is or mixtures thereof;

[0134] each LKb is independently L 2 — L 1 — B;

[0135] each B is independently a terminal group R 10 or a combination of 1), 2) and 3) below: 1) a self-cleaving spacer Sp1; 2) a bond or one or a combination of two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) a terminal group R 10 ;

[0136] R 10 is hydrogen or a group that can be left upon reaction with a group in the support;

[0137] each L 1 is independently a cleavable sequence 1 comprising an amino acid sequence that is cleavable by an enzyme, and the cleavable sequence 1 comprises 1-10 amino acids;

[0138] each L 2 is independently a bond; or C 2-20 alkylene, wherein one or more -CH2- structure in the alkylene is optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, -N⊕R 6 R 7 -, C 4-10 cycloalkylene, C 4-10 heterocyclylene, phenylene; wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from the group consisting of halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R9 ;

[0139] Ld2and each Ld1are independently a bond; or selected from -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-, or is a natural amino acid or an oligomeric natural amino acid having a degree of polymerization of 2-10, which natural amino acid or oligomeric natural amino acid is independently unsubstituted or substituted on the side chain by -(PEG) j -R 11 substituted;

[0140] -(PEG) t -, -(PEG) i - and -(PEG) j - are each a PEG fragment comprising the indicated number of consecutive -(O-C2H4)- or -(C2H4-O)- structural units, with optional additional C 1-10 alkylene;

[0141] 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

[0142] R 1 and R 2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group; or

[0143] R 3 and R 4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group;

[0144] R 11 is C 1-10 alkyl;

[0145] m is any integer from 1 to 3;

[0146] n is any integer from 2 to 20;

[0147] d is 0, or is any integer from 1 to 6;

[0148] Each i is an independent integer from 0 to 100, preferably 0 to 20; more preferably, each i is an independent integer from 0 to 12; more preferably 0 to 8; especially 4;

[0149] Each j is an independent integer from 1 to 100, preferably 1 to 20; preferably, each j is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12;

[0150] Each t is an independent integer from 1 to 100, preferably 1 to 20; more preferably, each t is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12.

[0151] In one implementation, L 2 Selected from: -(CH2) p -(CH2)2(CO)-, p is 0 or an integer from 1 to 5;

[0152]

[0153] b is an integer from 1 to 10.

[0154] In one implementation, p is 0 to 3; preferably 3.

[0155] In one implementation, L 2 Selected from: -(C2H4-O) p -(CH2)2(CO)-, where p is an integer from 1 to 5, more preferably p is 2 or 4.

[0156] In one implementation scheme, each of the above L 2 The carbonyl group in the structure and L 1 The connection is established, and another connection site is connected to opSu.

[0157] In one implementation scheme, each of the above L 2 The carbonyl group in the structure and L 1 One connection site is connected to the other, and another connection site is connected to the amide.

[0158] Ld2 and each Ld1 are independently keys or

[0159]

[0160] Each i is an independent integer between 0 and 100;

[0161] j and k are each an independent integer from 1 to 100.

[0162] In one implementation, each i is an integer independently from 0 to 20. In another implementation, each i is an integer independently from 0 to 12.

[0163] In one embodiment, j and k are each independently an integer from 1 to 20. In one embodiment, j and k are each independently an integer from 1 to 12.

[0164] In one embodiment, each i is independently an integer from 0 to 8; particularly 4.

[0165] In one embodiment, each j is independently an integer from 8 to 12; particularly 8 or 12.

[0166] In one embodiment, each k is independently an integer from 1 to 7; particularly 1 or 3 or 5.

[0167] In one embodiment, Ld2and each Ld1is independently a bond; or C 1-20 alkylene, or a PEG fragment of a particular length (denoted as -(PEG) i -) having an amino group and a carbonyl group at each of the two ends, or one or more natural amino acids independently unsubstituted or substituted with a PEG fragment of a particular length (denoted as -(PEG) j -) on a side chain.

[0168] In one embodiment, -(PEG) i- comprises -(O-C2H4) i - or -(C2H4-O) i -, and has an optional additional C 1-10 alkylene at one end; -(PEG) j comprises -(O-C2H4) j - or -(C2H4-O) j -, and has an optional additional C 1-10 alkylene at one end. In one embodiment, -(PEG) i comprises -C2H4-(O-C2H4) i - or -(C2H4-O) i -C2H4-.

[0169] It is understood that when two or more of the structures Ld1, B, L 2 or L 1 structures are present in a molecule, the respective structures of Ld1, B, L 2 or L 1 are independently selected. When two or more R x (x is 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) are present in a molecule, each R xare independently selected. In some embodiments, "x" in the molecule is denoted with or without an additional apostrophe (') or apostrophes (such as ", ", " ", etc.), e.g., R, R 1' , R 1” , R 1”' , R 2' , R 2” , R 2”' , etc., where each R x , with or without an additional apostrophe or apostrophes, is independently selected. Other R x , such as R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and "Ld1", "B", "L 2” ", and "L 1” " should be understood in a similar manner. In some embodiments, "i" in the molecule is denoted with or without an additional number, e.g., i1, i2, i3, i4, etc., where the number does not denote any order, but merely serves to distinguish "i". And each "i", with or without an additional number, is independently selected.

[0170] In one embodiment, cleavable sequence 1 is selected from the group consisting of 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; preferably, cleavable sequence 1 is Gly-Gly-Phe-Gly.

[0171] In one embodiment, W is hydrogen.

[0172] In one embodiment, W is -C2H4-(PEG) t -(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; in particular 8 or 12.

[0173] In one embodiment, R 11 is C 1-6 alkyl, preferably methyl.

[0174] In one embodiment, n is an integer from 2 to 5, in particular 3.

[0175] In one embodiment, d is 0, or any integer from 1 to 4; preferably 0, 1, 2, or 3.

[0176] Thiosuccinimides are unstable under physiological conditions and prone to undergo reverse Michael addition, leading to cleavage at the coupling site. In addition, thiosuccinimides can also undergo thiol exchange with other thiol compounds when present in the system. Both reactions can cause the detachment of the payload and lead to toxic side effects. In this context, when applied to linkers, the open ring succinimide structure no longer undergoes reverse Michael addition or thiol exchange, thus the product is more stable. The method of the ring opening reaction can be found in WO2015165413A1.

[0177] Compounds comprising open ring succinimide moieties can be purified by semi-preparative / preparative HPLC or other suitable separation means to obtain compounds with high purity and well-defined composition regardless of the efficiency of the succinimide ring opening reaction.

[0178] Moieties comprising a recognition sequence for a ligase acceptor or donor substrate

[0179] In one embodiment, G n is a recognition sequence of a ligase acceptor substrate that facilitates the enzyme-catalyzed coupling of the compound of formula (I) to a targeting molecule under the catalysis of a ligase. The targeting molecule is optionally modified and comprises a corresponding recognition sequence to the ligase acceptor substrate.

[0180] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of a natural transpeptidase, a non-natural transpeptidase, a variant thereof, and a combination thereof. The non-natural transpeptidase can be, but is not limited to, those obtained by engineering a natural transpeptidase. In a preferred embodiment, the ligase is selected from the group consisting of a natural sortase, a non-natural sortase, and a combination thereof. The classes of natural sortases include sortase A, sortase B, sortase C, sortase D, L. plantarum sortase, etc. (detailed description can be found in US20110321183A1, which is incorporated herein by reference). The type of ligase corresponds to the ligase recognition sequence and is thereby used to achieve specific coupling between different molecules or structural fragments.

[0181] In some embodiments, the ligase is a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, and L. plantarum sortase. In these embodiments, the recognition sequence of the ligase acceptor substrate is selected from the group consisting of oligomeric glycines, oligomeric alanines, and mixtures of oligomeric glycines / alanines with a degree of polymerization of 3-10. In a specific embodiment, the recognition sequence of the ligase acceptor substrate is G nwherein G is glycine (Gly) and n is an integer from 2 to 10.

[0182] In another specific embodiment, the ligase is sortase A from Staphylococcus aureus. Thus, the ligase recognition sequence can be the canonical recognition sequence for the enzyme as 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 acceptor substrate recognition sequence is G n wherein X can be any single amino acid, natural or unnatural; J is absent, or an amino acid fragment comprising 1-10 amino acids, optionally labeled. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment comprising 1 to 10 amino acids, wherein each amino acid is independently any natural or unnatural amino acid. In another embodiment, J is G m wherein m is an integer from 1 to 10. In yet another specific embodiment, the ligase donor substrate recognition sequence is LPETG (SEQ ID NO: 51). In another specific embodiment, the ligase donor substrate recognition sequence is LPETGG (SEQ ID NO: 52).

[0183] In one embodiment, the ligase is sortase B from Staphylococcus aureus, and the corresponding donor substrate recognition sequence can be NPQTN (SEQ ID NO: 53). In another embodiment, the ligase is sortase B from Bacillus anthracis, and the corresponding donor substrate recognition sequence can be NPKTG (SEQ ID NO: 54).

[0184] In yet another embodiment, the ligase is sortase A from Streptococcus pyogenes, and the corresponding donor substrate recognition sequence can be LPXTGJ, wherein J is as defined above. In another embodiment, the ligase is sortase subfamily 5 from Streptomyces coelicolor, and the corresponding donor substrate recognition sequence can be LAXTG (SEQ ID NO: 55).

[0185] In yet another embodiment, the ligase is sortase A from Lactobacillus plantarum, and the corresponding donor substrate recognition sequence can be LPQTSEQ (SEQ ID NO: 56).

[0186] The ligase recognition sequence can also be other brand new recognition sequences for transpeptidases that are optimized by artificial screening.

[0187] Moieties comprising a reactive group

[0188] Reactive groups for linking to a payload

[0189] In one embodiment, B is a terminal group R 10 and the cleavable sequence 1 in L 1 is linked to a payload. In this case, B is not present in the resulting molecule in which the cleavable sequence 1 is linked to a payload. In one embodiment, B is used to link a payload. To link to a payload, the compound of formula (I) comprises a reactive group. In one embodiment, B in the compound of formula (I) is linked to a payload via an amide bond or 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 (such as a reactive C=0 moiety, a reactive C=C-C=0 moiety, an amino group, an amine group, a hydroxyl group or a thiol group), or a reactive group for substitution reactions (such as a leaving group attached to an O, C, N or S atom). In one embodiment, the reactive group in B is independently selected from the group consisting of a carboxyl group, an active ester, an aldehyde group, an amino group, an amine group, a hydroxyl group and a thiol group. In a particular embodiment, the reactive group in B for linking to a payload is independently selected from the group consisting of an amino group, an amine group, a hydroxyl group, a thiol group, a carboxyl group and an active ester.

[0190] In one embodiment, the reactive group in B is independently an amino group, an amine group or a hydroxyl group, which reacts with a corresponding group in a payload, such as a carboxyl group, a sulfonic acid group, a phosphoryl group with a free -OH end, 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 reacts with a corresponding group in a payload, such as an amino group, an amine group or a hydroxyl group.

[0191] In one embodiment, the reactive group in B is independently an amino group, a hydroxyl group or a thiol group, which reacts with a corresponding group in a payload, such as a halogen, a hydroxyl group, an aldehyde group. In another embodiment, the reactive group in B is independently a hydroxyl group, which reacts with a corresponding group in a payload, such as a halogen or a hydroxyl group.

[0192] In one embodiment, each B is independently R 10or a combination of 1), 2) and 3) below: 1) a self-cleaving spacer Sp1; 2) a bond or one or a combination of two or more of the divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene and -(CO)-; and 3) a terminal group R 10 .

[0193] In one embodiment, Sp1 is selected from the group consisting of PABC, acetal, heteroacetal, and combinations thereof. In one embodiment, Sp1 is an acetal, a heteroacetal, or PABC. In one embodiment, the heteroacetal is selected from the group consisting of N,O-heteroacetals. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is attached to cleavable sequence 1; U is absent or is O, S, or NH, preferably O or S. In one embodiment, U is absent or is O, S, or NH, preferably O or S.

[0194] In one embodiment, B is R 10 , -NH-CH2-U-R 10 , -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 or -NH-CH2-U-(CH2) g -(CO)-R 10 .

[0195] In one embodiment, R 10 is hydrogen, hydroxyl, or In one embodiment, R 10 is hydrogen. In one embodiment, R 10 is hydroxyl or

[0196] In one embodiment, R 10 represents a moiety that will not appear in the product molecule resulting from the reaction of B with the payload.

[0197] Specific embodiments of compounds of formula (I)

[0198] In one embodiment, W is hydrogen, each LKa is In one embodiment, formula (I) has the structure of formula (I-a)

[0199]

[0200] In one embodiment, each LKa is In one embodiment, formula (I) has the structure of formula (I-b)

[0201]

[0202] In one embodiment, Ld2is a bond, and d is 0. In one embodiment, the compound of Formula (I-a) is:

[0203]

[0204] In one embodiment, d is 0, and Ld2is In one embodiment, the compound of Formula (I-a) is:

[0205]

[0206] In one embodiment, d is 1, 2, or 3, Ld2and each Ld1are independently selected from In one embodiment, the compound of Formula (I-a) is:

[0207]

[0208] In one embodiment, Ld2is d is 0. In one embodiment, the compound of Formula (I-a) is:

[0209]

[0210] In one embodiment, d is 1, 2, or 3, Ld2is and each Ld1is independently selected from In one embodiment, the compound of Formula (I-a) is:

[0211]

[0212] In one embodiment, d is 1, W is hydrogen, Ld2is and each Ld1is independently selected from In one embodiment, the compound of Formula (I-b) is:

[0213]

[0214] In one embodiment, d is 1, W is -C2H4-(PEG) t -C(O)NH2, Ld2is a bond, and each Ld1is independently selected from In one embodiment, the compound of Formula (I-b) is:

[0215]

[0216] In one embodiment, n is 3, L2 -(CH2)2(CO)-, p is 3, L p -(CH2)2(CO)-, p is 3, L 1 GGFG, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, I-a-0-1 has the structure:

[0217]

[0218]

[0219] In one embodiment, I-a-0-2 has the structure:

[0220] In one embodiment, I-a-0-3 has the structure:

[0221]

[0222]

[0223] In one embodiment, I-a-0-4 has the structure:

[0224]

[0225]

[0226] In one embodiment, I-a-0-5 has the structure:

[0227]

[0228]

[0229] In one embodiment, I-a-1-1 has the structure:

[0230] In one embodiment, I-a-1-2 has the structure:

[0231] In one embodiment, I-a-1-3 has the structure:

[0232]

[0233]

[0234] In one embodiment, I-a-1-4 has the structure:

[0235]

[0236]

[0237] In one embodiment, n is 3, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is GGFG, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, I-b-1 has the structure:

[0238]

[0239] In one embodiment, n is 3, L 2 is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is GGFG, B is -NH-CH2-U-R 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, g is 1. In one embodiment, I-b-0 has the structure:

[0240]

[0241] In one embodiment, i is 4, g is 1, R 11 is methyl.

[0242] In one embodiment, i is 2, g is 1, R 11 is methyl.

[0243] In one embodiment, I-a-0-2 is as follows (I-a-0-2-1 to I-a-0-2-3):

[0244]

[0245] In one embodiment, i is 4, j is 8, g is 1, R11 It is methyl. In one embodiment, Ia-1-2 is as follows (Ia-1-2-1 to Ia-1-2-3):

[0246]

[0247]

[0248] In one implementation, n is 3, i is 4, j is 12, g is 1, and R... 11 It is methyl. In one embodiment, Ia-1-2 is as follows (Ia-1-2-4 to Ia-1-2-6):

[0249]

[0250]

[0251] In one implementation, i is 4, j is 8, g is 1, m is 1, n is 3, and R 11 It is methyl. In one embodiment, Ib-1 is as follows (Ib-1-1 to Ib-1-3):

[0252]

[0253]

[0254] In one implementation, i is 4, j is 12, g is 1, m is 1, n is 3, and R 11 It is methyl. In one embodiment, Ib-1 is as follows (Ib-1-4 to Ib-1-1-6):

[0255]

[0256]

[0257] In one implementation, i is 4, j is 8, g is 1, m is 2, n is 3, and R 11 It is methyl. In one embodiment, Ib-1 is as follows (Ib-1-7 to Ib-1-9):

[0258]

[0259]

[0260] In one implementation, i is 4, j is 12, g is 1, m is 2, n is 3, and R... 11 It is methyl. In one embodiment, Ib-1 is as follows (Ib-1-10 to Ib-1-12):

[0261]

[0262]

[0263] 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, I-b-0 is as follows (I-b-0-1 to I-b-0-3):

[0264]

[0265]

[0266] 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, I-b-0 is as follows (I-b-0-4 to I-b-0-6):

[0267]

[0268]

[0269] 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, I-b-0 is as follows (I-b-0-7 to I-b-0-9):

[0270]

[0271]

[0272] 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, I-b-0 is as follows (I-b-0-10 to I-b-0-12):

[0273]

[0274]

[0275] Compound of formula (I) carrying a payload

[0276] B is covalently coupled to a support comprising a reactive group, to give a compound of formula (I) carrying a support.

[0277] In yet another aspect, there is provided a compound having the structure of formula (II)

[0278]

[0279] wherein

[0280] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2or LKb―P;

[0281] M is hydrogen or LKa-LKb―P;

[0282] with the proviso that Q and M are not simultaneously hydrogen;

[0283] P is a payload, said payload being attached to the B moiety or L 1 of the compound of formula (I);

[0284] n, d, Ld1, Ld2, t, LKaand LKbare as defined in formula (I).

[0285] As defined herein above, in the compound of formula (I), each LKbis independently L 2 -L 1 -B; each B is independently a terminal group R 10 or a combination of 1), 2) and 3) below: 1) a self-cleaving spacer Sp1; 2) a bond or a combination of one or two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) a terminal group R 10 ; R 10 is hydrogen or a group capable of leaving upon reaction with a group in the payload. In one embodiment, R 10 represents a moiety that will not be present in the product molecule resulting from the reaction of B with the payload.

[0286] In one embodiment, P is attached to the B moiety of the compound of formula (I) to form a compound of formula (II). As defined above, R 10 will not be present in the B―P structure of the compound of formula (II).

[0287] It is understood that when B in the compound of formula (I) is a terminal group R 10 , R 10 will not be present in the compound of formula (II). As a result, B is not present in the B―P structure of the compound of formula (II).

[0288] In one embodiment, M is hydrogen or LKa-L 2 ―L 1— B— P; wherein P is a moiety that is attached to the B portion of the compound of formula (I) or to L 1 a moiety that is attached to the B portion of the compound of formula (I) or to L 10 or a combination of 1), 2) and 3) below: 1) a self-excising spacer Sp1; 2) a bond or one or a combination of two or more of the divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) a terminal group R 10 ; R 10 is hydrogen or a group capable of leaving upon reaction with a group in the support; R 10 represents a moiety that will not be present in the product molecule resulting from the reaction of B with the support.

[0289] In one embodiment, M is hydrogen or LKa-L 2 — L 1 — B— P; wherein each B is independently absent, or a combination of 1) and 2) below: 1) a self-excising spacer Sp1; and 2) a bond or one or a combination of two or more of the divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-. In one preferred embodiment, M is hydrogen or LKa-L 2 — L 1 — B— P; wherein each B is independently absent, or is -NH-CH2-U- or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-. In another embodiment, M is hydrogen or LKa-L 2 — L 1 — B— P. In one embodiment, in LKa-L 2 — L 1 — B— P, B is absent. In one embodiment, LKa-L 2 — L 1 — B— P, B is a combination of 1) and 2) below: 1) a self-excising spacer Sp1; and 2) a bond or one or a combination of two or more of the divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10heterocyclyl and -(CO)-. In one embodiment, LKa-L 2 -L 1 In B―P, B is -NH-CH2-U- or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-; U is absent, or is O, S or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is linked to the support via an amide bond or an ester bond or an ether bond.

[0290] As defined herein above, when B in the compound of formula (I) is a terminal group R 10 B is not present in the B―P structure of the compound of formula (II). In this case, it is also understood that the cleavable sequence 1 in L 1 is linked to the support, forming the compound of formula (II), wherein B is not present in the resulting molecule where the cleavable sequence 1 is linked to the support. Thus, in one embodiment, P is linked to the L 1 part of the compound of formula (I), forming the compound of formula (II). Thus, in one embodiment, M is LKa-L 2 -L 1 -B―P, and B is not present; and M can also be represented as LKa-L 2 -L 1 -P.

[0291] Support

[0292] In the present context, the support can be selected from the group consisting of small molecule compounds, nucleic acids and analogues, tracer molecules (including fluorescent molecules and the like), short peptides, polypeptides, peptidomimetics and proteins. In one embodiment, the support is selected from the group consisting of small molecule compounds, nucleic acid molecules and tracer molecules. In a preferred embodiment, the support is selected from the group consisting of small molecule compounds. In a more preferred embodiment, the support is selected from the group consisting of cytotoxins and fragments thereof.

[0293] In one embodiment, the cytotoxin is selected from drugs targeting the microtubular cytoskeleton. In a preferred embodiment, the cytotoxin is selected from taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and derivatives thereof, indol-sulfonamides, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vindesine, deacetyl vinblastine, dolastatin 10 and analogs thereof, halichondrin B and eribulin, indol-3-oxoacetamides, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide. In another embodiment, the cytotoxin is selected from DNA topoisomerase inhibitors, such as camptothecin and derivatives thereof, mitoxantrone, mitoguazone. In a preferred embodiment, the cytotoxin is selected from nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenamet, phenesterine, prednimustine, trofosfamide, uracil mustard. In yet another preferred embodiment, the cytotoxin is selected from nitrosoureas, such as carmustine, fotemustine, lomustine, nimustine, ranimustine. In one embodiment, the cytotoxin is selected from aziridines. In a preferred embodiment, the cytotoxin is selected from benzodopa, carboquone, meturedopa, and uredopa. In one embodiment, the cytotoxin is selected from anti-tumor antibiotics. In a preferred embodiment, the cytotoxin is selected from enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from dynemicin, esperamicin, neocarzinostatin, and aclacinomycin. In another preferred embodiment, the cytotoxin is selected from actinomycin, anthramycin, bleomycin, calicheamicin, carabicin, carminomycin, and cardinophyllin, carminomycin, dactinomycin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, nogalamycin, olivomycins, peplomycin, poptidmycin, puromycin, ferric adriamycin, zorubicin. In yet another preferred embodiment, the cytotoxin is selected from a sesquiterpene. In a more preferred embodiment, the cytotoxin is selected from T-2 toxin, verracurin A, roridin A, and snake venom. In one embodiment, the cytotoxin is selected from anti-tumor amino acid derivatives.In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, diazomycin, 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 folic acid, methotrexate, pteropterin, trimetrexate, and edatrexate. In an embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiamiprine, thioguanine. In yet another embodiment, the cytotoxin is selected from the group consisting of 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, floxuridine. In an embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of calusterone, dromostanolone propionate, epitiostanol, megestrol, testolactone. In another embodiment, the cytotoxin is selected from the group consisting of antiadrenals. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In an embodiment, the cytotoxin is selected from the group consisting of antiandrogens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate, and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors. In another embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicine, taxoids, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmucin, SN-38, a cryptophycin analog, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastatins, and pyrrolobenzodiazepine. In a specific embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicine, taxoids, auristatins, and maytansinoids.

[0294] In a specific embodiment, the cytotoxin is duocuritinib or a derivative thereof, such as DX8951f, etc.

[0295] In another specific embodiment, the cytotoxin is a maytansinoid, such as DM1, etc. Note that when a cytotoxin comprising a thiol moiety is used, the thiol moiety is capable of reacting with a maleimide moiety to form a thiosuccinimide (e.g., maytansinoid, e.g., DM1), and the cytotoxin can be directly linked via the thiosuccinimide. In this case, it will be understood that in some embodiments the payload and the thiol moiety together constitute the cytotoxin, and thus in this case the payload represents the remainder of the cytotoxin molecule other than the thiol moiety.

[0296] In one embodiment, the cytotoxin is a maytansinoid, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), and the like. Synthesis and structure of maytansinoid compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0297] The support contains reactive groups which can react with the reactive groups in the compound of formula (I) and thus covalently couple the support to the compound of formula (I). Compounds which do not contain reactive groups need to be appropriately derivatized to obtain a support.

[0298] In one embodiment, the cytotoxin is a compound of formula (i)

[0299]

[0300] wherein,

[0301] a* is 0 or 1 ;

[0302] The carbon atoms marked with p1*and p2*are each asymmetric centers and the asymmetric centers are in S-configuration, R-configuration or racemic;

[0303] L 1* is selected from C 1-6 alkylene, the C 1-6 alkylene is unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2;

[0304] M* is -CH2-, -NH- or -O-;

[0305] L 2* is C 1-3 alkylene;

[0306] R 1* and R 2* are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0307] In one embodiment, the cytotoxin is a compound of formula (i')

[0308]

[0309] wherein, g is any integer from 1 to 6.

[0310] In one embodiment, g* is any integer from 1 to 3, preferably 1.

[0311] In one embodiment, L 1*selected from C 1-6 straight-chain alkylene, C 1-6 branched-chain alkylene, C 3-6 cyclic alkylene and C 3-4 cyclic alkyl-C 1-2 straight-chain alkylene groups, each independently unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2. In one embodiment, L 1* selected from C 1-4 alkylene, said C 1-4 alkylene is unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2. In one preferred embodiment, L 1* selected from -CH2-, -C2H4-, or each independently substituted by at least one substituent selected from the group consisting of halogen, -OH and -NH2. In one preferred embodiment, L 1* selected from -CH2-, wherein the position of attachment to the carbonyl group is marked by “#”. In one more preferred embodiment, L 1* selected from -CH2-, wherein the position of attachment to the carbonyl group is marked by “#”. In one particular embodiment, L 1* selected from -CH2-, wherein the position of attachment to the carbonyl group is marked by “#”. In one preferred embodiment, halogen is selected from F, Cl and Br, in particular F.

[0312] In one embodiment, a* is 1, M* is -CH2-, -NH- or -O-; and L 2* -C2H4-. In another embodiment, a* is 1, M* is -CH2- and L 2* -CH2-. In one embodiment, a* is 0.

[0313] In one embodiment, the carbon atom marked with p1* is in S configuration or racemic, preferably in S configuration. In another embodiment, the carbon atom marked with p2* is in S configuration or racemic, preferably in S configuration.

[0314] In one embodiment, R 1* and R 2* are each independently selected from the group consisting of hydrogen, C 1-3 alkyl, halogen or C 1-3 alkoxy. In one preferred embodiment, R 1* and R 2* are each independently selected from the group consisting of CH3-, F, Cl, Br and CH3O-. In one embodiment, R 1*selected from CH3- or Cl. In another embodiment, R 2* is F.

[0315] In one embodiment, a* is 0, L 1* is selected from -CH2-, wherein "#" marks the position of attachment to the carbonyl group. In one embodiment, a* is 1, L 1* is M* is O, and L 2* is -C2H4-.

[0316] In one embodiment, a* is 0, R 1* is Cl, R 2* is F, and L 1* is selected from -CH2-, In one embodiment, a* is 0, R 1* is CH3-, R 2* is F, and L 1* is selected from wherein "#" marks the position of attachment to the carbonyl group.

[0317] In one embodiment, a* is 1, R 1* is CH3-, R 2* is F, L 1* is M is O, and L 2* is -C2H4-.

[0318] In one embodiment, the cytotoxin is selected from the following compounds; wherein the wavy bond indicates the site of attachment to the compound of formula (I).

[0319]

[0320] In some embodiments, the payload is selected from DX8951f (compound 9), DXd-(1) (compound 10), DXd-(2) (compound 14), preferably DX8951f, DXd-(1), more preferably DXd-(1), most preferably

[0321] Preparation of a compound of formula (I) carrying a payload

[0322] In one embodiment, the linking unit and the payload are linked via a reactive group as defined above using any reaction known in the art, including but not limited to condensation reactions, nucleophilic addition, electrophilic addition, and the like.

[0323] In one embodiment, the payload is a cytotoxin. In one embodiment, the linker-payload intermediate (numbered LBx) is as shown in the table below.

[0324]

[0325]

[0326] *: n is 3 for all linkers listed.

[0327] Conjugates and their preparation

[0328] In addition, a compound of Formula (I) with a payload comprising a portion of a ligase recognition sequence can be coupled to other molecules comprising a ligase recognition sequence, and thus can be used, for example, to make targeted molecule-drug conjugates, such as antibody-drug conjugates. Thus, in yet another aspect, there is provided a conjugate comprising a compound of Formula (I), a targeting molecule, and a payload.

[0329] In yet another aspect, there is provided a conjugate having the structure of Formula (III):

[0330]

[0331] wherein,

[0332] n, d, Ld1, and Ld2 are as defined in Formula (I);

[0333] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2, or LKb— P;

[0334] M is hydrogen or LKa-LKb— P;

[0335] with the proviso that Q and M are not both hydrogen;

[0336] P is a payload, said payload being attached to the B portion of the compound of Formula (I) or to L 1 ;

[0337] A is an anti-Trop2 antibody or antigen binding fragment thereof attached to the G n portion of the compound of Formula (I); G is glycine;

[0338] z is an integer from 1 to 20.

[0339] In one embodiment, LKaand LKbare as defined in Formula (I).

[0340] In one embodiment, the conjugate has an integer or non-integer drug antibody ratio (DAR) of 1 to 20.

[0341] As defined above, in one embodiment, G n is a recognition sequence of a ligase acceptor substrate that facilitates the enzyme-catalyzed coupling of the compound of formula (I) to a targeting molecule under catalysis of a ligase. The targeting molecule is optionally modified and comprises a corresponding recognition sequence of a ligase acceptor or donor substrate.

[0342] It is understood that when the antibody (or antigen binding fragment) is coupled to G n of the compound of formula (I) under catalysis of a ligase, the recognition sequence of the ligase acceptor substrate and the ligase donor substrate recognition sequence react with each other and form the resulting sequence. n

[0343] In one embodiment, the antibody (or antigen binding fragment) comprises LPXTGJ, which is a ligase donor substrate recognition sequence, wherein J is as defined above. When coupled to G n (which is a corresponding recognition sequence of a ligase acceptor substrate), the peptide bond upstream of the glycine in the LPXTGJ sequence is cleaved by sortase A and the resulting intermediate is linked to the free N-terminus of G n to create a new peptide bond. The resulting sequence is LPXTG n (SEQ ID NO: 57). The sequence G n and LPXTGJ are as defined above.

[0344] In one embodiment, P is linked to the B portion or L 1 portion of the compound of formula (I), A is linked to the G n portion of the compound of formula (I) to form a compound of formula (III).

[0345] As defined above, R 10 does not appear in the B―P structure of the compound of formula (III). As defined above, when B is a terminal group R 10 in the compound of formula (I), B is not present in the B―P structure of the compound of formula (III).

[0346] As defined above, in the A-G n structure of the compound of formula (III), A optionally comprises a corresponding sequence resulting from the reaction of a recognition sequence of a ligase acceptor substrate and a ligase donor substrate recognition sequence.

[0347] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein P is a payload linked to the B portion or L 1 portion of the compound of formula (I); each B is independently a terminal group R 10or a combination of 1), 2) and 3) below: 1) a self-elimination spacer Sp1; 2) a bond or a combination of one or two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) a terminal group R 10 ; R 10 is hydrogen or a group capable of leaving upon reaction with a group in the support; R 10 represents a structural moiety which is not present in the product molecule resulting from the reaction of B with the support.

[0348] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or a combination of 1) and 2) below: 1) a self-elimination spacer Sp1; and 2) a bond or a combination of one or two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-. In one preferred embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or -NH-CH2-U- or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-. In another embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P. In one embodiment, in LKa-L 2 ―L 1 ―B―P, B is absent. In one embodiment, LKa-L 2 ―L 1 ―B―P, B is a combination of 1) and 2) below: 1) a self-elimination spacer Sp1; and 2) a bond or a combination of one or two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-. In one embodiment, LKa-L 2 ―L 1- B - P, B is -NH-CH2-U- or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, U is absent, or is O, S or NH, preferably O or S. In one embodiment, B in the compound of formula (I) is attached to the support via an amide bond or an ester bond or an ether bond. In one embodiment, M is LKa-L 2 - L 1 - B -, B is absent; and M can also be represented as LKa-L 2 - L 1 - P.

[0349] Targeting molecules

[0350] In one embodiment, the targeting molecule is an anti-Trop2 antibody or antigen binding fragment thereof comprising a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein,

[0351] the V H comprises:

[0352] (i) HCDR1 comprising the amino acid sequence of X1X2GMX3 (SEQ ID No: 1), wherein X1 is N, T or A, X2 is Y or A, X3 is N or Q;

[0353] (ii) HCDR2 comprising the amino acid sequence of WINTX4X5GX6PX7YX8X9DFKG (SEQ ID NO: 2), wherein, 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, X9 is D or E;

[0354] (iii) HCDR3 comprising the amino acid sequence of X 10 GFGSSYWYFDV (SEQ ID NO: 3), wherein X 10 is G or S;

[0355] and / or

[0356] the V L comprises:

[0357] (i) LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0358] (ii) LCDR2, which comprises the amino acid sequence of SASYRYT (SEQ ID NO: 15); and

[0359] (iii) LCDR3, which comprises the amino acid sequence of QQHYITPLT (SEQ ID NO: 16).

[0360] In one embodiment, HCDR1 comprises 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).

[0361] In one embodiment, HCDR2 comprises the amino acid of WINTYTGEPTYTDDFKG (SEQ ID NO: 8), WINTHSGVPKYAEDFKG (SEQ ID NO: 9), WINTDSGEPTYTDDFKG (SEQ ID NO: 10).

[0362] In one embodiment, HCDR3 comprises the amino acid of GGFGSSYWYFDV (SEQ ID NO: 11) or SGFGSSYWYFDV (SEQ ID NO: 12).

[0363] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), wherein

[0364] the V H comprises:

[0365] (i) HCDR1, which comprises the amino acid sequence of X1AGMN, wherein X1 is N or A;

[0366] (ii) HCDR2, which comprises the amino acid sequence of WINTDSGEPTYTDDFKG (SEQ ID NO: 10);

[0367] (iii) HCDR3, which comprises the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 11); and / or

[0368] the V L comprises:

[0369] (i) LCDR1, which comprises the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0370] (ii) LCDR2, which comprises the amino acid sequence of SASYRYT (SEQ ID NO: 15);

[0371] (iii) LCDR3, which comprises the amino acid sequence of QQHYITPLT (SEQ ID NO: 16).

[0372] In one embodiment, the V H comprises:

[0373] (i) HCDR1, which comprises the amino acid sequence of SEQ ID NO: 4,

[0374] (ii) HCDR2, which comprises the amino acid sequence of SEQ ID NO: 8, and (iii) HCDR3, which comprises the amino acid sequence of SEQ ID NO: 11; and / or

[0375] the V L comprises:

[0376] (i) LCDR1, which comprises the amino acid sequence of SEQ ID NO: 13,

[0377] (ii) LCDR2, which comprises the amino acid sequence of SEQ ID NO: 15, and (iii) LCDR3, which comprises the amino acid sequence of SEQ ID NO: 16.

[0378] In one embodiment, the V H comprises:

[0379] (i) HCDR1, which comprises the amino acid sequence of SEQ ID NO: 5,

[0380] (ii) HCDR2, which comprises the amino acid sequence of SEQ ID NO: 9, and (iii) HCDR3, which comprises the amino acid sequence of SEQ ID NO: 12; and / or

[0381] the V L comprises:

[0382] (i) LCDR1, which comprises the amino acid sequence of SEQ ID NO: 14,

[0383] (ii) LCDR2, comprising the amino acid sequence of SEQ ID NO: 15, and (iii) LCDR3, comprising the amino acid sequence of SEQ ID NO: 16.

[0384] In one embodiment, the V H comprising:

[0385] (i) HCDR1, comprising the amino acid sequence of SEQ ID NO: 4,

[0386] (ii) HCDR2, comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HCDR3, comprising the amino acid sequence of SEQ ID NO: 11; and / or

[0387] In one embodiment, the V L comprising:

[0388] (i) LCDR1, comprising the amino acid sequence of SEQ ID NO: 13,

[0389] (ii) LCDR2, comprising the amino acid sequence of SEQ ID NO: 15, and (iii) LCDR3, comprising the amino acid sequence of SEQ ID NO: 16.

[0390] In one embodiment, the V H comprising:

[0391] (i) HCDR1, comprising the amino acid sequence of SEQ ID NO: 7,

[0392] (ii) HCDR2, comprising the amino acid sequence of SEQ ID NO: 10, and (iii) HCDR3, comprising the amino acid sequence of SEQ ID NO: 11; and / or

[0393] In one embodiment, the V L comprising:

[0394] (i) LCDR1, comprising the amino acid sequence of SEQ ID NO: 13,

[0395] (ii) LCDR2, comprising the amino acid sequence of SEQ ID NO: 15, and (iii) LCDR3, comprising the amino acid sequence of SEQ ID NO: 16.

[0396] In one embodiment, the VH comprises:

[0397] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 6,

[0398] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and

[0399] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0400] and / or

[0401] the V L comprises:

[0402] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0403] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0404] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0405] In one embodiment, the V H comprises the structure: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4, FR1 comprises the amino acids of SEQ ID NO: 17, FR2 comprises the amino acids of SEQ ID NO: 18, FR3 comprises the amino acids of SEQ ID NO: 19, and FR4 comprises the amino acids of SEQ ID NO: 20.

[0406] In one embodiment, the V H comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 21-25. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 21. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 22. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 23. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 24. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 25.

[0407] In one embodiment, the V L comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27. In one embodiment, the V L comprises the amino acid sequence of SEQ ID NO: 26. In one embodiment, the V L comprises the amino acid sequence of SEQ ID NO: 27.

[0408] In one embodiment, the V H comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24; and / or the V L comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

[0409] In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 21, the V L comprises the amino acid sequence of SEQ ID NO: 26. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 22, the V L comprises the amino acid sequence of SEQ ID NO: 27. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 23, the V L comprises the amino acid sequence of SEQ ID NO: 26. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 24, the V L comprises the amino acid sequence of SEQ ID NO: 26. In one embodiment, the V H comprises the amino acid sequence of SEQ ID NO: 25, the V L comprises the amino acid sequence of SEQ ID NO: 26.

[0410] In one embodiment, the antibody or antigen binding fragment comprises: a heavy chain constant domain (CH) comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28; and / or

[0411] a light chain constant domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0412] In one embodiment, the antibody or antigen-binding fragment comprises: a heavy chain constant domain (CH) comprising the amino acid sequence of SEQ ID NO: 28, and

[0413] a light chain constant domain comprising the amino acid sequence of SEQ ID NO: 29.

[0414] In one embodiment, the antibody or antigen-binding fragment binds TROP2 with an equilibrium dissociation constant (K D ) of about 0.5 nM to about 20 nM. In one embodiment, the K D value is about 0.5 nM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 15 nM, about 18 nM, about 20 nM, or a range between any two values inclusive of the end point values. In one embodiment, the K D value is about 7.5 nM to about 13.5 nM.

[0415] In one embodiment, the targeting molecule is an anti-human TROP2 antibody or antigen-binding fragment thereof.

[0416] In one embodiment, the antibody is a recombinant antibody selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, an antibody fragment, and an antibody mimetic. In one embodiment, the antibody mimetic is selected from the group consisting of a scFv, a minibody, a diabody, a nanobody.

[0417] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30-33, and / or a light chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35.

[0418] 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 inclusive of the end point values.

[0419] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30-33, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35.

[0420] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0421] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0422] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0423] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0424] Examples of anti-human TROP2 antibodies include, but are not limited to, Trodelvy antibody (hRS7) and DS1062 antibody (Datopotamab). Antibody sequences are shown in Table 1.

[0425] In one embodiment, the pharmaceutical combination comprises an antibody or antigen-binding fragment.

[0426] Table 1 Antibody Sequences

[0427]

[0428]

[0429]

[0430] Antibodies conjugated to a linker-payload

[0431] For conjugation with the compound of Formula (I), the antibody herein can comprise a modified moiety to link to Gn in the compound of Formula (I). The position of introduction of such modified moiety is not limited, for example, it can be introduced at the C-terminus or N-terminus of the heavy chain or light chain of the antibody, but is not limited thereto.

[0432] In one embodiment, the conjugate herein can specifically bind to TROP2 on the surface of tumor cells and selectively kill tumor cells expressing TROP2, the conjugate is formed by conjugating an anti-human TROP2 antibody and a payload. In another preferred embodiment, there is provided a use of the conjugate herein or the pharmaceutical combination herein in the manufacture of a medicament for treating a disease, disorder, or condition selected from TROP2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from breast cancer, urothelial cancer, lung cancer, liver cancer, endometrial cancer, head and neck cancer, ovarian cancer, and the like.

[0433] In an alternative embodiment, the modified moiety for conjugation with Gn in the compound of Formula (I) can be introduced to a non-terminal position of the heavy or light chain of the antibody using, for example, chemical modification methods.

[0434] In one embodiment, the targeting molecule herein is an antibody or an antigen binding fragment thereof, which can comprise a terminal modification. The terminal modification refers to a modification at the C- or N-terminus of the heavy or light chain of the antibody, for example, the terminal modification comprises a ligase recognition sequence. In another embodiment, the terminal modification can further comprise a spacer Sp2containing 2-100 amino acids, wherein the antibody, Sp2, and the ligase recognition sequence are connected in sequence. In a preferred embodiment, Sp2is a spacer sequence containing 2-20 amino acids. In a particular embodiment, Sp2is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS, in particular, GA.

[0435] In a preferred embodiment, the light chain of the antibody or antigen binding fragment thereof comprises three types: wild type (LC); C-terminal modified light chain (LCCT) modified by direct introduction of ligase recognition sequence LPXTG; and C-terminal modified light chain (LCCT L ) modified by introduction of short peptide spacer plus ligase donor substrate recognition sequence LPXTG. The heavy chain of the antibody or antigen binding fragment thereof comprises three types: wild type (HC); C-terminal modified heavy chain (HCCT) modified by direct introduction of ligase recognition sequence LPXTG; and C-terminal modified heavy chain (HCCT L ) modified by introduction of short peptide spacer plus ligase donor substrate recognition sequence LPXTG. X can be any single amino acid, either natural or unnatural. In one embodiment, X is glycine. The modified antibody sequences are shown in Table 2.

[0436] The conjugate herein can further comprise a payload. The payload is as described above.

[0437] In one embodiment, the pharmaceutical combination comprises an antibody-drug conjugate (ADC).

[0438] Table 2 Modified antibody sequences

[0439]

[0440]

[0441] Specific embodiments of conjugates

[0442] In one embodiment, Q is hydrogen, each LKa is In one embodiment, Formula (III) has the structure of Formula (III-a):

[0443]

[0444] wherein A is an anti-TROP2 antibody or antigen binding fragment, and the antibody or antigen binding fragment is modified to conjugate to (Gly) n

[0445] In one embodiment, Ld2 is a bond, d is 0. In one embodiment, the compound of Formula (III-a) is as follows:

[0446]

[0447] In one embodiment, d is 0, Ld2 is In one embodiment, the compound of Formula (III-a) is as follows:

[0448]

[0449] In one embodiment, d is 1, 2, or 3, Ld2 and each Ld1 are independently selected from In one embodiment, the compound of Formula (III-a) is as follows:

[0450]

[0451] In one embodiment, Ld2 is d is 0. In one embodiment, the compound of Formula (III-a) is as follows:

[0452]

[0453] In one embodiment, d is 1, 2, or 3, Ld2 is and each Ld1 is independently selected from In one embodiment, the compound of Formula (III-a) is as follows:

[0454] ​

[0455]

[0456] 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, 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.

[0457] In one embodiment, LKa is In one embodiment, formula (III) has the structure of formula (III-b):

[0458]

[0459] wherein A is an anti-TROP2 antibody or antigen binding fragment, which is modified to have the structure of (Gly) n linked.

[0460] In one embodiment, Q is hydrogen, d is 1, Ld2 is and Ld1 is selected from In one embodiment, the compound of formula (III-b) is as follows:

[0461]

[0462] In one embodiment, Q is -C2H4-(PEG) t -(CO)NH2, d is 1, Ld2 is a bond, and Ld1 is selected from In one embodiment, the compound of formula (III-b) is as follows:

[0463]

[0464] 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.

[0465] In one embodiment, B in the compound of formula (I) is a terminal group R 10 and L 1wherein B is absent in the resulting molecule where the cleavable sequence 1 is linked to the payload. In this case, M can be understood as LKa-L 2 — L 1 — B— P, wherein B is absent. In this case, M can also be represented as LKa-L 2 — L 1 — P. In one embodiment, n is 3, L 2 is -(CH2) p — (CH2)2(CO)—, p is 3, L 1 is GGFG, B is -NH-CH2-U- or is absent or is -NH-CH2-U-(CR 1 R 2 ) g — (CO)—, U is O, g is 1.

[0466] In one embodiment, conjugate III-a-0-1 has the structure:

[0467]

[0468] wherein A is an anti-TROP2 antibody or antigen binding fragment that is modified to be linked to (Gly) n .

[0469] In one embodiment, conjugate III-a-0-2 has the structure:

[0470]

[0471] wherein A is an anti-TROP2 antibody or antigen binding fragment that is modified to be linked to (Gly) n .

[0472] In one embodiment, conjugate III-a-0-3 has the structure:

[0473] wherein A is an anti-TROP2 antibody or antigen binding fragment that is modified to be linked to (Gly) n .

[0474] In one embodiment, conjugate III-a-0-4 has the structure:

[0475]

[0476] wherein A is an anti-TROP2 antibody or antigen binding fragment that is modified to be linked to (Gly)n is connected.

[0477] In one embodiment, conjugate III-a-0-5 has the structure:

[0478]

[0479] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is connected to (Gly) n is connected.

[0480] In one embodiment, conjugate III-a-1-1 has the structure:

[0481]

[0482] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is connected to (Gly) n is connected.

[0483] In one embodiment, conjugate III-a-1-2 has the structure:

[0484]

[0485] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is connected to (Gly) n is connected.

[0486] In one embodiment, conjugate III-a-1-3 has the structure:

[0487]

[0488] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is connected to (Gly) n is connected.

[0489] In one embodiment, conjugate III-a-1-4 has the structure:

[0490]

[0491] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is connected to (Gly) n is connected.

[0492] In one embodiment, i is 4, g is 1, R 11 is methyl.

[0493] In one embodiment, n is 3, L 2is -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is GGFG, B is -NH-CH2-U-, U is O. In one embodiment, conjugate III-b-1-1 has the following structure:

[0494]

[0495] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is attached to (Gly) n .

[0496] In one embodiment, conjugate III-a-0-2 is as follows (conjugate III-a-0-2-1):

[0497]

[0498] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is attached to (Gly) n .

[0499] In one embodiment, i is 4, j is 8, g is 1, R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-1):

[0500]

[0501]

[0502] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is attached to (Gly) n .

[0503] In one embodiment, i is 4, j is 12, g is 1, R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-1):

[0504]

[0505] wherein A is an anti-TROP2 antibody or antigen binding fragment that, upon modification, is attached to (Gly) n .

[0506] In one embodiment, i is 4, j is 12, R 11is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-2):

[0507]

[0508] wherein A is an anti-TROP2 antibody or antigen binding fragment, which antibody or antigen binding fragment, after modification, is linked to (Gly) n .

[0509] In one embodiment, i is 4, j is 12, m is 1, and R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-2):

[0510]

[0511] wherein A is an anti-TROP2 antibody or antigen binding fragment, which antibody or antigen binding fragment, after modification, is linked to (Gly) n .

[0512] In one embodiment, n is 3, i is 4, j is 12, m is 1, and R 11 is methyl. In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-2):

[0513]

[0514] wherein A is an anti-TROP2 antibody or antigen binding fragment, which antibody or antigen binding fragment, after modification, is linked to (Gly) n .

[0515] Preparation of conjugates

[0516] The conjugates herein can be prepared by any method known in the art. In some embodiments, the conjugates are prepared by a ligase-catalyzed site-specific conjugation method, in which a targeting molecule modified by a ligase recognition sequence is conjugated to a compound of Formula (I) carrying a payload. The method comprises steps A and B.

[0517] Step A. Preparation of a Linker Unit-Payload Intermediate

[0518] In a preferred embodiment, B in the compound of Formula (I) is covalently linked to a payload containing another reactive group via a reactive group.

[0519] The conjugate intermediate prepared using the compound of formula (I) herein has a well-defined structure, well-defined composition and high purity, thus introducing less or no additional impurities when the coupling reaction is performed with the antibody. When this intermediate is used for ligation enzyme catalyzed site-specific conjugation with a modified antibody containing a ligation enzyme recognition sequence, a highly quality-controllable homogeneous ADC is obtained.

[0520] Step B. Linking the targeting molecule to the compound of formula (I) carrying a payload

[0521] The targeting molecule herein can be coupled to the compound of formula (I) carrying a payload (i.e. compound of formula (II)) by any method known in the art.

[0522] The targeting molecule and the compound of formula (I) carrying a payload are linked to each other via a ligation enzyme specific substrate recognition sequence. The recognition sequence depends on the specific ligation enzyme used. In one embodiment, the targeting molecule is an antibody with a terminal modification based on the recognition sequence introduced at the C-terminus of the light chain and / or the heavy chain, and the targeting molecule is coupled to the compound of formula (II) under catalysis of a wild-type or optimized engineered ligation enzyme or any combination thereof and under suitable catalytic reaction conditions.

[0523] In one particular embodiment, the ligation enzyme is sortase A, and the coupling reaction can be represented by the following scheme:

[0524]

[0525] The triangle represents a portion of the antibody; the pentagon represents a portion of the compound of formula (II). n, X and J are defined as above. When coupled to G n The peptide bond upstream of the glycine in the LPXTGJ sequence is cleaved by sortase A, and the resulting intermediate is linked to the free N-terminus of G n to create a new peptide bond. The resulting amino acid sequence is LPXTG n . The sequence G n and LPXTGJ are defined as above.

[0526] Metabolism of the conjugate in a physiological environment

[0527] When part or all of the linker is cleaved in the tumor cell, the anti-tumor compound moiety is released to exert the anti-tumor effect of the anti-tumor compound. When the linker is cleaved at the site of the linkage to the drug, the anti-tumor compound is released in its native structure to exert its native anti-tumor effect.

[0528] In one embodiment, cleavable sequence 1 (such as GGFG) can be cleaved by lysosomal enzymes (such as Cathepsin B and / or Cathepsin L).

[0529] In one embodiment, Sp1 comprises a self-cleaving spacer. In one embodiment, Sp1 comprises a PABC, acetal, or heteroacetal. In one embodiment, L 1 GGFG. In one embodiment, the linker comprises -GGFG-NH-CH2-O-. In one embodiment, -GGFG-NH-CH2-O- represents a combination of a restriction enzyme cleavage site and a self-cleaving spacer that will be cleaved and release a molecule of interest (such as a drug) in a cell.

[0530] Pharmaceutical combinations and pharmaceutical formulations

[0531] It is a further object herein to provide a pharmaceutical combination comprising a prophylactically or therapeutically effective amount of a conjugate herein and an anti-PD-1 antibody, wherein the conjugate has the structure of Formula (III):

[0532]

[0533] wherein,

[0534] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2, or LKb―P;

[0535] M is hydrogen or LKa-LKb―P; wherein

[0536] each LKa is independently selected from

[0537] opSu is or mixtures thereof;

[0538] each LKb is independently L 2 ―L 1 ―B;

[0539] each B is independently absent, or a combination of 1) a self-cleaving spacer Sp1; and 2) a bond or one or a combination of two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclyl, and -(CO)-; preferably, B is -NH-CH2-U- or is absent or -NH-CH2-U-(CR 1 R 2 ) g-(CO)-; U is not present, or is O, S or NH, preferably O or S;

[0540] with the proviso that Q and M are not simultaneously hydrogen;

[0541] P is a support, which is attached to the B moiety of formula (III) or L 1 ;

[0542] each L 1 is independently a cleavable sequence 1 comprising an amino acid sequence which is cleavable by an enzyme, and the cleavable sequence 1 comprises 1-10 amino acids;

[0543] each L 2 is independently a bond; or C 2-20 alkylene, wherein one or more -CH2- structures in said alkylene are optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, -N 6 R 7 -, C 4-10 ycloalkylene, C 4-10 heterocyclylene, phenylene; wherein said cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from the group consisting of halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 and -C 1-10 alkylene-O-R 9 ;

[0544] Ld2and each Ld1are independently a bond; or selected from the group consisting of -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-, or a natural amino acid or an oligomeric natural amino acid having a degree of polymerization of 2-10, independently unsubstituted or substituted in the side chain by -(PEG) j -R 11 ;

[0545] -(PEG)t-, -(PEG)i- and -(PEG)j- are each a PEG fragment comprising the specified number of consecutive -(O-C2H4)- or -(C2H4-O)- structural units with an optional additional C 1-10 alkylene at one end;

[0546] R 1 , R 2 , R3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 each independently is selected from hydrogen, halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, C 4-10 cycloalkyl; or

[0547] R 1 and R 2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group; or

[0548] R 3 and R 4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group;

[0549] R 11 is C 1-10 alkyl;

[0550] m is any integer from 1 to 3;

[0551] n is any integer from 2 to 20;

[0552] d is 0, or is any integer from 1 to 6;

[0553] each i is independently an integer from 0 to 100, preferably from 0 to 20; preferably, each i is independently an integer from 0 to 12; more preferably from 0 to 8; especially 4;

[0554] 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; especially 8 or 12;

[0555] 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; especially 8 or 12;

[0556] A is an anti-TROP2 antibody or antigen binding fragment thereof, preferably after modification to be linked to the Gn moiety in formula (III), and G is glycine;

[0557] z is an integer from 1 to 20.

[0558] In some embodiments, wherein the conjugate has the structure of formula (III-a) or formula (III-b):

[0559]

[0560] In some embodiments, wherein the conjugate has the following structure:

[0561]

[0562]

[0563]

[0564]

[0565] Preferably, z is 1 to 4; preferably 2;

[0566] i, i1, i2, i3, i4 are each independently an integer from 0 to 100, preferably from 0 to 20; preferably, i, i1, i2, i3, i4 are each independently an integer from 0 to 12; more preferably from 0 to 8; in particular 4;

[0567] 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; in particular 8 or 12;

[0568] Preferably, n is 3, L2 is -(CH2) p -(CH2)2(CO)- or -(C2H4-O) p -(CH2)2(CO)-, p is 2 to 4, L1 is Gly-Gly-Phe-Gly, B is -NH-CH2-U- or is absent or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, U is absent or U is O, g is 1;

[0569] 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; in particular 8 or 12;

[0570] m is any integer from 1 to 3; in particular 1 or 2.

[0571] In some embodiments, wherein the payload is a cytotoxin or fragment thereof, optionally derivatized to link to the B moiety or L1 moiety in the compound of formula (III);

[0572] Preferably, the cytotoxin is selected from taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and derivatives thereof, indol-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, leurosidine, vindesine, deoxyvinblastine, dolastatin 10 and analogs thereof, halichondrin B, eribulin, indol-3-oxoacetamides, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, leurosidine, camptothecin and derivatives thereof, mitoxantrone, mitoguazone, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carbinoquinone, demetozole, uramustine, dianemycin, esperamicins, neocarzinostatin, aclacinomycin, actinomycin, antinomycin, bleomycin, bleomycin C, calicheamicin, carminomycin, carzinophilin, carminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, idarubicin, marcellomycin, mitomycin, noranomycin, olivomycin, peplomycin, porfiromycin, puromycin, ranomycin, rodorubicin, streptonigrin, streptozocin, zinostatin, zorubicin, trichothecenes, T-2 toxin, veracin A, myriocin, ophiocordin, ubenimex, diazomycin, 6-diazo-5-oxo-L-norleucine, methotrexate, pemetrexed, trimetrexate, edatrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, megestrol, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprolide acetate, a protein kinase inhibitor, and a proteasome inhibitor; and / or

[0573] is selected from vinca alkaloids, colchicine, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmycin, SN-38, amanitin analogs, deruxitecan, duocarmycin, calicheamicin, spongistatin, dolastatin, pyrrolobenzodiazepine is selected from auristatins, in particular MMAE, MMAF, or MMAD; and / or

[0574] is selected from auristatins, in particular MMAE, MMAF, or MMAD; and / or

[0575] is selected from auristatins, in particular MMAE, MMAF, or MMAD; and / or

[0576] In some embodiments, the payload has the structure of Formula (i):

[0577]

[0578] wherein

[0579] a* is 0 or 1 ;

[0580] the carbon atoms marked with p1* and p2* are each a center of asymmetry and the center of asymmetry is in the S configuration, the R configuration or racemic;

[0581] L 1* is selected from C 1-6 alkylene, said C 1-6 alkylene is unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2;

[0582] M* is -CH2-, -NH- or -O-;

[0583] L 2* is C 1-3 alkylene;

[0584] R 1* and R 2* are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, halogen and C 1-6 alkoxy.

[0585] In some embodiments, wherein L 1* is selected from C 1-6 linear alkylene, C 1-6 branched alkylene, C 3-6 cyclic alkylene and C 3-4 cyclic alkyl-C 1-2 linear alkylene groups, wherein the alkylene and cyclic alkylene are each independently unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2; preferably L 1* is selected from C 1-4 alkylene, wherein the alkylene is unsubstituted or substituted by one substituent selected from the group consisting of halogen, -OH and -NH2; more preferably L 1* is selected from -CH2-, -C2H4-, or each independently substituted by at least one substituent selected from the group consisting of halogen, -OH and -NH2; most preferably L 1* is selected from -CH2-, wherein the position of attachment to the carbonyl group is marked with "#".

[0586] In some embodiments, wherein a* is 0.

[0587] In some embodiments, wherein R 1* is selected from C 1-6 alkyl, halogen; preferably R 1* is methyl or CI.

[0588] In some embodiments, wherein R 2* is selected from C 1-6 alkyl, halogen; preferably, R 2* is F.

[0589] In some embodiments, wherein the payload is selected from

[0590]

[0591] In particular, is selected from

[0592]

[0593] In some embodiments, wherein the conjugate is selected from

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601] each g is independently an integer from 1 to 6, preferably from 1 to 3; more preferably 1;

[0602] R 1 and R 2 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 3-6 membered cycloalkyl group; preferably R 1 and R 2 are hydrogen;

[0603] 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; in particular 8 or 12;

[0604] m is any integer from 1 to 3; in particular 1 or 2;

[0605] z is an integer from 1 to 20; in particular 2 or 4, more preferably 2.

[0606] In some embodiments, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) H ) and a light chain variable region (VL) L , wherein

[0607] the VHcomprises:

[0608] (i) HCDR1 comprising the amino acid sequence of X1X2GMX3 (SEQ ID No: 1), wherein X1is N, T or A, X2is Y or A, X3is N or Q;

[0609] (ii) HCDR2 comprising the amino acid sequence of WINTX4X5GX6PX7YX8X9DFKG (SEQ ID NO: 2), wherein, X4is Y, H or D, X5is T or S, X6is E or V, X7is T or K, X8is T or A, X9is D or E;

[0610] (iii) HCDR3 comprising the amino acid sequence of X 10 GFGSSYWYFDV (SEQ ID NO: 3), wherein X 10 is G or S;

[0611] and / or

[0612] the VLcomprises:

[0613] (i) LCDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0614] (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).

[0615] In some embodiments, wherein,

[0616] the VH H comprises:

[0617] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4,

[0618] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0619] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 12;

[0620] and / or

[0621] said V L comprises:

[0622] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0623] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0624] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; or

[0625] said V H comprises:

[0626] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 5,

[0627] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and

[0628] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 12;

[0629] and / or

[0630] said V L comprises:

[0631] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 14,

[0632] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0633] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 16;

[0634] or

[0635] said V H comprises:

[0636] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4,

[0637] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and

[0638] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0639] and / or the V L comprises:

[0640] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0641] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0642] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 16;

[0643] or

[0644] the V H comprises:

[0645] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 7,

[0646] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and

[0647] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0648] and / or

[0649] the V L comprises:

[0650] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0651] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0652] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 16;

[0653] or

[0654] the V H comprises:

[0655] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 6,

[0656] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and

[0657] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11 ;

[0658] and / or

[0659] the V L comprises:

[0660] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0661] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0662] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0663] In some embodiments, wherein the V H comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 21 to 25, and / or

[0664] the V L comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27.

[0665] In some embodiments, wherein the antibody or antigen binding fragment comprises: a heavy chain constant domain (CH) comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 28; and / or

[0666] a light chain constant domain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0667] In some embodiments, wherein the antibody or antigen binding fragment binds TROP2 with an equilibrium dissociation constant (K D ) of about 0.5 nM to about 20 nM.

[0668] In some embodiments, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30 to 33, and / or a light chain comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35.

[0669] In some embodiments, wherein the antibody or antigen-binding fragment comprises a C-terminal modification of the heavy chain and / or a C-terminal modification of the light chain, such that the antibody, Sp2 and a ligase donor substrate recognition sequence are connected in sequence; Sp2 is a spacer selected from the group consisting of GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS; the ligase donor substrate recognition sequence is LPXTGJ, wherein X can be any single amino acid, natural or unnatural; and J is absent or an amino acid fragment comprising 1-10 amino acids.

[0670] In some embodiments, wherein the modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 30 to 33, and / or a light chain of SEQ ID NO: 40 or SEQ ID NO: 41.

[0671] or

[0672] the modified antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO: 36 to 39, and / or a light chain of SEQ ID NO: 34 or SEQ ID NO: 35.

[0673] In some embodiments, wherein the conjugate has an integer or non-integer drug to antibody ratio (DAR) of 1 to 19.

[0674] In some embodiments, wherein the anti-PD-1 antibody is a mouse antibody, a humanized antibody or a fully human antibody. In some embodiments, wherein 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, and does not bind to mouse FGFR3.

[0675] In some embodiments, wherein the anti-PD-1 antibody is selected from the group consisting of: pembrolizumab, nivolumab, tremelimumab, tislelizumab, sintilimab and camrelizumab.

[0676] In some embodiments, the pharmaceutical combination optionally further comprises a pharmaceutically acceptable carrier.

[0677] The pharmaceutical combinations herein can be administered in any form that achieves the intended prophylactic, palliative, prophylactic, or curative effect in humans or animals. For example, various suitable dosage forms can be prepared depending on the route of administration, particularly injections, such as lyophilized powders for injection, injections, or sterile powders for injection.

[0678] The term "pharmaceutically acceptable" means that which is acceptable to the patient's tissues, when applied to the tissues of a patient, does not produce an adverse, allergic or other untoward reaction, etc., commensurate with a reasonable benefit / risk ratio, and is effective for its intended use.

[0679] The term "pharmaceutically acceptable carrier" refers to those carriers that are pharmaceutically acceptable and do not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline and the like. Pharmaceutically acceptable carriers also include carrier materials that render the composition amenable to physiological conditions, such as pH adjusters, buffers, toxicity adjusters, and the like, as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. In some embodiments, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active ingredient for treatment is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and solutions of glucose in water or glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetate, citrate or phosphate, if desired.

[0680] In one embodiment, the drug antibody ratio (DAR) of the conjugates herein is an integer or non-integer from about 1 to about 20, such as 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, about 1 to about 2. In a particular embodiment, the DAR of the conjugates herein is about 2, about 4, about 6, or about 8.

[0681] A kit comprising the pharmaceutical combination.

[0682] In some embodiments, the kit comprises:

[0683] a first packaging unit comprising the conjugate,

[0684] a second packaging unit comprising an anti-PD-1 antibody; and

[0685] optionally, instructions for administering the conjugate and the anti-PD-1 antibody to a subject.

[0686] Methods of treatment and uses

[0687] A pharmaceutical combination comprising the conjugate and the anti-PD-1 antibody or a kit comprising the conjugate and the anti-PD-1 antibody is used for treating a tumor and / or an autoimmune disease. Tumors susceptible to treatment by the conjugate include those characterized by expression of specific tumor-associated antigens or cell surface receptors, and these tumors will be recognized by the targeting molecule in the conjugate and can be killed by the payload / cytotoxin in the conjugate.

[0688] Thus, in yet another aspect, there is also provided the use of a pharmaceutical combination or kit herein in the manufacture of a medicament for the treatment of a disease, disorder, or condition selected from a tumor or an autoimmune disease.

[0689] In another aspect, there is provided a pharmaceutical combination or kit herein for use in the treatment of a tumor or an autoimmune disease.

[0690] In another aspect, there is provided a pharmaceutical combination or kit herein for use in the treatment of a tumor or an autoimmune disease.

[0691] In yet another aspect, there is provided a method of treating a tumor or an autoimmune disease, the method comprising administering to an individual in need thereof an effective amount of a pharmaceutical combination or kit herein.

[0692] In yet another aspect, there is provided a method of treating a subject having a cancer or reducing the likelihood of progression of a cancer, the method comprising administering to the subject an effective amount of a conjugate having the structure of Formula (III), and administering to the subject an effective amount of an anti-PD-1 antibody.

[0693] In some embodiments, wherein the conjugate has the structure of Formula (III):

[0694]

[0695] wherein,

[0696] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2or LKa-LKb-P;

[0697] M is hydrogen or LKa-LKb-P; wherein

[0698] each LKa is independently selected from

[0699] opSu is or mixtures thereof;

[0700] each LKb is independently L 2 — L 1 — B;

[0701] each B is independently absent, or a combination of 1) a self-cleaving spacer Sp1; and 2) a bond or one or a combination of two or more divalent groups selected from the group consisting of -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and -(CO)-; preferably B is -NH-CH2-U- or is absent or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-; U is absent, or is O, S or NH, preferably O or S;

[0702] with the proviso that Q and M are not simultaneously hydrogen;

[0703] P is a payload, said payload being linked to the B moiety or L 1 of formula (III);

[0704] each L 1 is independently a cleavable sequence 1 comprising an amino acid sequence that is cleavable by an enzyme, and said cleavable sequence 1 comprises 1-10 amino acids;

[0705] each L 2 is independently a bond; or C 2-20 alkylene, wherein one or more -CH2- structure in said alkylene is optionally replaced by -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -, -N⊕R 6 R 7 -, C 4-10 cycloalkylene, C 4-10 heterocyclylene, phenylene; wherein said cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted with at least one substituent selected from the group consisting of halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, -C 1-10 alkylene-NH-R 8 , and -C 1-10 alkylene-O-R 9 ;

[0706] Ld2and each Ld1is independently a bond; or selected from -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-, or is a natural amino acid or an oligomeric natural amino acid having a degree of polymerization of 2-10, which natural amino acid or oligomeric natural amino acid is independently unsubstituted or substituted on the side chain by -(PEG) j -R 11 substituted;

[0707] -(PEG) t -, -(PEG) i -, and -(PEG) j each is a PEG fragment comprising the indicated number of consecutive -(O-C2H4)- or -(C2H4-O)- structural units, with optional additional C 1-10 alkylene;

[0708] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 each independently is selected from hydrogen, halogen, -C 1-10 alkyl, -C 1-10 haloalkyl, C 4-10 cycloalkylene; or

[0709] R 1 and R 2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group; or

[0710] R 3 and R 4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkyl group;

[0711] R 11 is C 1-10 alkyl;

[0712] m is any integer from 1 to 3;

[0713] n is any integer from 2 to 20;

[0714] d is 0, or is any integer from 1 to 6;

[0715] 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; in particular 4;

[0716] 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; in particular 8 or 12;

[0717] 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; in particular 8 or 12;

[0718] A is an anti-TROP2 antibody or an antigen binding fragment thereof, preferably modified to be linked to the Gn moiety in formula (III), and G is glycine;

[0719] z is an integer from 1 to 20.

[0720] In some embodiments, the conjugate has the structure of formula (III-a) or formula (III-b) below.

[0721] In some embodiments, the conjugate has the structure of formula (III-a) or formula (III-b) below.

[0722] 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;

[0723] Preferably, z is from 1 to 4; preferably 2;

[0724] i, i1, i2, i3, i4 are each independently an integer from 0 to 100, preferably from 0 to 20; preferably, i, i1, i2, i3, i4 are each independently an integer from 0 to 12; more preferably from 0 to 8; in particular 4;

[0725] 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; in particular 8 or 12;

[0726] Preferably, n is 3, L 2 is -(CH2) p -(CH2)2(CO)- or -(C2H4-O) p -(CH2)2(CO)-, p is from 2 to 4, L1 is Gly-Gly-Phe-Gly, B is -NH-CH2-U- or is absent or is -NH-CH2-U-(CR1R2) g -(CO)-, U is absent or U is O, g is 1;

[0727] each t is independently an integer from 1 to 100, preferably 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably 8 to 12; in particular 8 or 12;

[0728] m is any integer from 1 to 3; in particular 1 or 2.

[0729] In some embodiments, wherein the payload is a cytotoxin or fragment thereof, optionally derivatized to attach to the B moiety or L 1 moiety in the compound of formula (III).

[0730] In some embodiments, wherein the cancer overexpresses TROP2 or the cancer has a TROP2 gene mutation.

[0731] In some embodiments, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

[0732] In some embodiments, wherein the anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody; and / or

[0733] 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.

[0734] In some embodiments, the method of any one of claims 30 to 32, wherein the anti-PD-1 antibody is selected from the group consisting of: pembrolizumab, nivolumab, tepilumab, tislelizumab, sintyrozumab, and camrelizumab.

[0735] In some embodiments, wherein the conjugate is or the conjugate is ADC-2.

[0736] In some embodiments, wherein the conjugate and the anti-PD-1 antibody are administered simultaneously as part of the same pharmaceutical formulation.

[0737] In some embodiments, wherein the conjugate and the anti-PD-1 antibody are administered simultaneously as part of different pharmaceutical formulations.

[0738] In some embodiments, wherein the conjugate and the anti-PD-1 antibody are administered at different times.

[0739] In yet another aspect, there is provided the use of an effective amount of a conjugate and an effective amount of an anti-PD-1 antibody in the manufacture of a medicament for treating a subject having a cancer.

[0740] In some embodiments, wherein the cancer overexpresses TROP2 or the cancer has a TROP2 gene mutation.

[0741] In some embodiments, wherein the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, pharyngeal squamous cell carcinoma, and urothelial carcinoma.

[0742] In some embodiments, wherein the anti-PD-1 antibody is a mouse antibody, a humanized antibody, or a fully human antibody; and / or

[0743] 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 does not bind to mouse FGFR3.

[0744] In some embodiments, wherein the anti-PD-1 antibody is selected from the group consisting of: pembrolizumab, nivolumab, tepilumab, tislelizumab, sintilimab, and camrelizumab.

[0745] In some embodiments, wherein the conjugate is or the conjugate is ADC-2.

[0746] In some embodiments, wherein the conjugate and the anti-PD-1 antibody are administered simultaneously as part of the same pharmaceutical formulation.

[0747] In some embodiments, wherein the conjugate and the anti-PD-1 antibody are administered simultaneously as part of different pharmaceutical formulations.

[0748] In some embodiments, wherein the conjugate and the anti-PD-1 antibody are administered at different times.

[0749] In a preferred embodiment, the conjugate herein can specifically bind to TROP2 on the surface of tumor cells and selectively kill tumor cells expressing TROP2, the conjugate is formed by conjugating an anti-human TROP2 antibody and a small molecule cytotoxin. In another preferred embodiment, there is provided a use of the conjugate (or antibody) herein or the pharmaceutical combination herein in the manufacture of a medicament for treating a disease, disorder, or condition selected from the group consisting of TROP2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is a TROP2-positive tumor. In one embodiment, the TROP2-positive tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, colon cancer, pharyngeal squamous cell carcinoma, urothelial carcinoma, and the like.

[0750] The dose of the conjugate (or antibody) administered to a subject can be adjusted to a considerable extent. The dose can vary depending on the specific administration route and the needs of the subject, and can be judged by a healthcare professional.

[0751] Beneficial effects

[0752] The antibody-drug conjugate of the present application uses a specially designed linker-load, has higher stability, and can achieve excellent efficacy at a lower DAR, thereby reducing side effects and improving the therapeutic index.

[0753] The linker unit with a unique structure is used herein, and a ligase is used to catalyze the coupling of the targeting molecule and the load. The conjugate herein has good uniformity, high activity, and high selectivity. In addition, the toxicity of the linker-load intermediate is much lower than that of the free load, so the harmfulness of the drug manufacturing process is smaller, which is beneficial to industrial production.

[0754] The conjugate herein achieves at least one of the following technical effects:

[0755] (1) High inhibitory activity on target cells, or strong killing effect on target cells.

[0756] (2) Good physicochemical properties (e.g., solubility, physical and / or chemical stability).

[0757] (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life, and duration of action).

[0758] (4) Good safety (low toxicity to non-target normal cells or tissues, and / or fewer side effects, wider therapeutic window), etc.

[0759] (5) Highly modular design, simple assembly of multiple drugs.

[0760] Examples

[0761] Preparation Examples

[0762] In order to make the purpose and technical solutions clearer, the present application is further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present application. The specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0763] Instruments, materials and reagents

[0764] Unless otherwise specified, the instruments and reagents used in the examples are commercially available. The reagents can be used directly without further purification.

[0765] MS: Thermo Fisher Q Exactive Plus, Water 2795-Quattro Micro Triple Quadrupole Mass Spectrometer

[0766] HPLC: Waters 2695, Agilent 1100, Agilent 1200

[0767] Semi-preparative HPLC: Lisure HP plus 50D

[0768] Flow cytometry: CytoFLEX S

[0769] HIC-HPLC: Butyl-HIC; mobile phase A: 25 mM PB, 2 M (NH4)2S04, pH 7.0; mobile phase B: 25 mM PB, pH 7.0; flow rate: 0.8 mL / min; collection time: 25 min; injection volume: 20 pg; column temperature: 25 °C; detection wavelength: 280 nm; sample compartment temperature: 8 °C.

[0770] SEC-HPLC: Column: TSK-gel G3000 SWXL, TOSOH 7.8 mm ID x 300 mm, 5 pm; mobile phase: 0.2 M KH2P04, 0.25 M KC1, pH 6.2; flow rate: 0.5 mL / min; collection time: 30 min; injection volume: 50 pL; column temperature: 25 °C; detection wavelength: 280 nm; sample tray temperature: 8 °C.

[0771] CHO was obtained from Thermo Fisher Scientific; pcDNA3.3 was obtained from Life Technology; HEK293F was obtained from Prejin; PEI MAX transfection reagent was obtained from Polyscience; MabSelect Sure Pro A was obtained from GE; Capto SimpAct 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; Capan-1 was obtained from ATCC CAT# CRL-1573; The optimized recombinant enzyme sortase A was prepared in E. coli, which was derived from Staphylococcus aureus.

[0772] Example 1 Construction of antibody expression vector, antibody expression, purification and identification

[0773] 1.1 Construction of expression vector encoding anti-TROP2 antibody

[0774] To generate the expression vector encoding the light chain of anti-TROP2 antibody, the nucleic acid sequence of LC was cloned into pCDNA3.3 vector (Life technology) alone; to generate the expression vector encoding the heavy chain of anti-TROP2 antibody, the nucleic acid sequence of HC was cloned into pCDNA3.3 vector (Life technology) alone.

[0775] Table 3 Antibody sequence

[0776]

[0777] Note: In the preparation of ADC, the peptide bond upstream of GG in the LPETGG sequence is cleaved by sortase A, and the resulting intermediate is connected to the free N-terminal of G3 in the linker-load, resulting in a new peptide bond.

[0778] 1.2 Expression of anti-TROP2 antibody

[0779] Plasmids encoding light and heavy chains of anti-TROP2 antibodies were paired and mixed at a mass ratio of 2: 1. The plasmid pairs were mixed with PEI MAX (Polyscience) transfection reagent after being diluted with HEK293F base medium separately. The mixture was left at room temperature and added to HEK293F seed cell culture solution. After the cells were cultured at 32°C for 24 hours, sampling was performed for cell density and viability analysis, and 10% volume of HEK293F feed medium was added. Then the culture temperature was adjusted to 32°C for subsequent culture. At 72 hours of incubation, the cell culture was sampled again for cell density and viability analysis. At 144 hours of incubation, the cell culture was sampled for cell density and viability analysis.

[0780] 1.3 Purification of anti-TROP2 antibodies

[0781] 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 a buffer of 50 mM Tris, 150 mM NaCl, pH 7.4. The supernatant of the cell culture was then obtained and loaded onto the column. The column was washed with a buffer of 50 mM Tris, 150 mM NaCl, pH 7.4 to remove non-specifically bound proteins. The antibodies were then eluted with a 50 mM citrate buffer (pH 3.5) and the antibody-containing eluate was adjusted to pH 6.5 with 1 M Tris-HCl (pH 9.0). Finally, the buffer of the antibodies was exchanged to a buffer of 50 mM Tris, 150 mM NaCl, pH 7.4 by Anicon Ultra-15 centrifugal filters (Merk Millipore).

[0782] 1.4 Binding kinetics and affinity analysis

[0783] Binding kinetics and affinity analysis were performed. Surface plasmon resonance (SPR) analysis was performed on a Biacore T200 (GE healthcare) with a sensor chip protein A (GE healthcare) according to the manufacturer's instructions. All measurements were performed at 25°C in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P20). About 110 RU-140 RU (resonance units) of each purified antibody was captured onto flow cell 2 and flow cell 4 (i.e. the reaction surface) of the sensor chip, respectively. HBS-EP buffer was used to record the baseline before the injection of the analyte. The analyte was injected at a flow rate of 30 μL / min. The dissociation phase was recorded for 5 minutes. The sensor chip was regenerated with 10 mM glycine, pH 1.5. The data were analyzed using the Biacore T200 evaluation software. + buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P20). About 110 RU-140 RU (resonance units) of each purified antibody was captured onto flow cell 2 and flow cell 4 (i.e. the reaction surface) of the sensor chip, respectively. HBS-EP +Buffered to serve as reference surface.

[0784] In HBS-EP + Serial dilutions of recombinant extracellular domain of human TROP2 (i.e. analyte) (Acrobio Systems) were prepared in buffer (243 nM, 81 nM, 27 nM, 9 nM, 3 nM, 1 nM, 0.333 nM and 0.111 nM, respectively). After antibody capture, serial dilutions of TROP2 were injected in sequence, with an injection time of three minutes (association phase) at a flow rate of 30 pL / min, followed by a ten-minute buffer flow (dissociation phase). The chip surface was regenerated by two 30-second pulses of 10 mM glycine-HCl, pH 1.5, at a flow rate of 50 pL / min. The collected data were processed on Biacore T200 Evaluation software using methods known in the art, including the following steps: (1) zeroing the y-axis response and x-axis injection start, (2) double referencing, first subtracting the reference surface data from the reaction surface data to obtain analyte injection curves, and then subtracting the buffer injection curves from the analyte injection curves, and (3) kinetic analysis with a 1 : 1 binding model fit. The results for each antibody were expressed as Ka (association rate), Kd (dissociation rate) and K D (equilibrium dissociation constant).

[0785] 1.5 SEC-HPLC detection of anti-TROP2 antibodies

[0786] Antibody samples were centrifuged at 12,000 rpm for 5 minutes and the supernatant was loaded onto the SEC-HPLC column to detect the percentage of monomeric (corresponding to intact antibody), high molecular weight (HMW, corresponding to antibody aggregates resulting from aggregation) and low molecular weight (LMW, corresponding to antibody fragments resulting from degradation) forms of each antibody.

[0787]

[0788] Trop2 is widely expressed in normal tissues, and the affinity of Ab13 and Ab16 is reduced. Antibodies with low affinity should be able to improve safety while maintaining the efficacy that has been validated in previous experiments.

[0789] 1.6 Internalization activity

[0790] MDA-MB-468 with good viability were collected after trypsinization, suspended in cold FACS buffer (DPBS + 2% FBS) and adjusted to 2 x 10 6cells / mL. Anti-Trop2 antibody and isotype control antibody samples were mixed and incubated with anti-human IgG-Fc-AF647 secondary antibody at a 1 : 1 molar ratio for 20 minutes at room temperature for fluorescent labeling. The labeled antibodies were added to the cell suspension at a final concentration of 10 pg / mL. The antibody-cell mixture was incubated on ice for one 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 antibody internalization for 0, 10, 30, 60, 90, 120, 180, and 240 minutes, and 1 x 105 5 cells / well were transferred to a V-bottom 96-well plate. Cells were returned to ice bath to terminate internalization. Non-internalized cells (0 min) were divided into MAX and MIN groups, while antibody-internalized cells were labeled as Group I. The MIN and I groups were washed twice with quenching buffer (150 mM NaCl + 100 mM glycine, pH = 2.0 to 2.5) to dissociate surface-bound antibodies. After quenching, all groups were washed twice with FACS buffer and analyzed by flow cytometry in the APC channel.

[0791] MFI data were plugged into the following equation and analyzed by one-phase exponential association function in Prism 6.

[0792] Isotype control: MFI (I) = MFI (样品，I) - MFI (同型对照抗体，I)

[0793] MFI (MAX) = MFI (样品，MAX) - MFI (阴性抗体，MAX)

[0794] MFI (MIN) = MFI (样品，MIN) - MFI (阴性抗体，MIN)

[0795] Internalization ratio: R (t) = (MFI (I) - MFI (MIN) ) / MFI (MAX) x 100%.

[0796] As shown in Figure 1.1 , all four antibodies showed comparable internalization activity against MDA-MB-468.

[0797] Preparation of intermediates of Example 2

[0798] Preparation of Linker-Support 1 and Linker-Support 2 of Example 2.1

[0799]

[0800] opSu is a mixture of

[0801] Preparation of intermediate MC-GGFG-DXd

[0802] Intermediate MC-GGFG-DXd can be commercially available or prepared according to the procedure described in EP2907824. This compound is used to prepare Linker- support 1.

[0803] Preparation of Linker-support intermediate 1

[0804]

[0805] Linker-support intermediate 1 can be synthesized by conventional solid-phase polypeptide synthesis using Rink-amide-MBHA-resin. Fmoc is used to protect the amino acids in the linking unit. The coupling reagent is selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the product is cleaved from the resin using a TFA / TIS / H20 solution. The product is purified by preparative HPLC, lyophilized and stored for use. MS m / z: [M-H] - = 1382.6.

[0806] Preparation of Linker-support 1

[0807] Linker-support intermediate 1 and MC-GGFG-DXd (molar ratio of about 1:2) are weighed out, dissolved in water and DMF respectively, and then mixed thoroughly to obtain a mixture, and reacted at 0-40°C for 0.5-30 hours. After the reaction is completed, an appropriate amount of Tris base solution or other solution to promote ring-opening reaction is directly added to the reaction mixture, and the reaction is continued at 0-40°C for 0.2-20 hours. After the reaction is completed, the product is purified by semi-preparative / preparative HPLC, and then lyophilized to obtain Linker-support 1. MS m / z: [(M+3H) / 3] + = 1163.3.

[0808] Preparation of Linker-support 2

[0809] The following linker-support (Linker-support 2) can be prepared using a similar synthetic route and reagents as Linker-support 1.

[0810]

[0811] opSu is a mixture of

[0812] Example 2.2 Preparation of Linker-support 3 and Linker-support 4​​

[0813] Preparation of Intermediate 11

[0814]

[0815] Step A: N-(2-bromo-5-fluorophenyl)acetamide: To a stirred solution of acetic anhydride (214 g, 2.10 mol) in acetic acid (500 mL) was added concentrated H2SO4(3 mL) followed by the portionwise addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) at room temperature. The mixture was stirred for 3 h, then poured into 2000 mL of ice water. A precipitate formed, which was collected by filtration and dried under vacuum at room temperature to give N-(2-bromo-5-fluorophenyl)acetamide (105 g) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.68 (dd, J = 8.9, 6.0 Hz, 1H), 7.61 (ddd, J = 10.7, 5.3, 3.1 Hz, 1H), 7.02 (ddd, J = 8.9, 8.0, 3.1 Hz, 1H), 2.11 (s, 3H). MS m / z 232.0 (M+H).

[0816] Step B: N-(5-fluoro-2-(l-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL) was added dropwise n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) at -78 °C over 1 h. After completion, the mixture was stirred under N2for 0.5 h. Then a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise at -78 °C over 0.5 h, and the mixture was stirred at -78 °C to room temperature for 6 h. The mixture was poured into 500 mL of saturated aqueous NH4Cl solution at 0 °C. It was extracted with ethyl acetate (500 mL x 3), washed with brine (250 mL x 2), dried over Na2SO4, and concentrated. The mixture was triturated with (PE / EA = 1:1, 100 mL) for 10 min, filtered, the filter cake was collected and dried under vacuum to give N-(5-fluoro-2-(l-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. LCMS m / z 206.1 (M-18+H), 246.1 (M+Na).

[0817] Step C: N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l-yl)acetamide: To a stirred mixture of N-(5-fluoro-2-(l-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) in CH2Cl2(170 mL) and water (170 mL) was added silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) and the mixture was stirred at 30 °C for 6 h. The mixture was filtered over celite and washed with CH2Cl2(100 mL), the filtrate was concentrated and purified by FCC (EA / PE = 0% - 40%) to give N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l-yl)acetamide (14 g) as a light yellow solid. MS m / z 222.1 (M+H).

[0818] Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-l- yl)acetamide: To a stirred mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l- yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) was added 1-nitroso butane (8.48 g, 63.28 mmol) at 0 °C, followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0 °C for 2 h. Upon completion, the mixture was acidified by HC1 (2 N) to adjust pH = 3. The mixture was extracted by ethyl acetate (200 mL x 3), washed by brine (100 mL x 2), dried over Na2SO4and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 min, filtered, the filter cake was collected and dried under vacuum to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-l-yl)acetamide (12 g) as a yellow solid. MS m / z 251.1 (M+H).

[0819] Step E: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l,7-diyl)diacetamide: To a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-l- yl)acetamide (12 g, 47.96 mmol) in acetic anhydride (90 mL) and THF (90 mL) was added 10% Pd / C (1 g), the mixture was stirred at 25 °C for 16 h under H2 atmosphere. After cooling to 0 °C, Et3N (20 mL) was added dropwise, the mixture was stirred at 0 °C for 1 h. Filtration on celite, the filtrate was poured into ice water (500 mL). Extraction with ethyl acetate (500 mL x 3), washed with brine (250 mL x 2), dried over Na2S04, and concentrated. The residue was triturated with t-butyl methyl ether (120 mL) for 10 min, filtered, the filter cake was collected and dried under vacuum to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l,7-diyl)diacetamide (7.9 g) as a yellow solid. MS m / z 279.1 (M+H).

[0820] Step F: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l,7-diyl)diacetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l,7-diyl)diacetamide (7.9 g, 28.39 mmol) in MeOH (150 mL) was added aqueous HC1 (2 N, 150 mL), the mixture was stirred at 50 °C for 7 h. After cooling to 0 °C, saturated aqueous NaHC03was added dropwise to adjust pH = 8. Extraction with ethyl acetate (200 mL x 3), washed with brine (200 mL x 2), dried over Na2S04and concentrated under reduced pressure to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l,7-diyl)diacetamide (6.0 g) as a yellow solid. 1 HNMR (400 MHz, Chloroform-d) δ 6.57 (s, 3H), 6.18 (td, J = 11.1, 2.4 Hz, 2H), 4.52 (dt, J = 13.3, 5.0 Hz, 1H), 3.13 (ddd, J = 17.5, 13.0, 4.6 Hz, 1H), 3.00 - 2.81 (m, 1H), 2.69 (dtd, J = 9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J = 13.0, 4.3 Hz, 1H). MS m / z 237.1 (M+H).

[0821] Step G: N-(8-amino-5-chloro-6-fluoro-l-oxo-l,2,3,4-tetrahydronaphthalen-2- yl)acetamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-l,7- diyl)diacetamide (4.0 g, 16.93 mmol) in DMF (80 mL) was added NCS (2.26 g, 16.93 mmol) in portions 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 give N-(8-amino-5-chloro-6-fluoro-l-oxo-l,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).

[0822] Step H: N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15- hexahydro-lH, 12H-benzo[de]pyranopyrrolo[3',4':6,7]indolizino[l,2-b]quinolin-l- yl)acetamide: To a mixture of N-(8-amino-5-chloro-6-fluoro-l-oxo-l,2,3,4- tetrahydronaphthalen-2-yl)acetamide (4.0 g, 14.78 mmol) in toluene (400 mL) was added (S)-4-ethyl-4-hydroxy-7,8-dihydro-lH-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridine p-toluenesulfonate (1.11 g, 4.43 mmol) and o-cresol (10 mL), the mixture was heated under reflux under N2for 24 hours. The solvent was removed by reduced pressure, and the mixture was purified by FCC (THF / CH2Cl2= 0% - 60%) to give N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15- hexahydro-lH, 12H-benzo[de]pyranopyrrolo[3',4':6,7]indolizino[l,2-b]quinolin-l- yl)acetamide (4.1 g) as a brown solid. MS m / z 498.1 (M+H).

[0823] Step I: (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13- dione: A mixture of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of aqueous concentrated HCl was stirred at 70 °C for 36 h. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid. MS (ESI) m / z 456.1 (M+H). 2 Step I: (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13- dione: A mixture of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 1-yl)acetamide (2.0 g, 4.02 mmol) in 20 mL of aqueous concentrated HCl was stirred at 70 °C for 36 h. The mixture was concentrated under reduced pressure to give crude (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15- hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (2 g) as a brown solid. MS (ESI) m / z 456.1 (M+H).

[0824] Preparation of Intermediate 12 (12-1, 12-2)

[0825]

[0826] Intermediate 12 (12-1, 12-2) was prepared by preparative HPLC from (9S)-1- amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (Intermediate 11) as a TFA salt.

[0827]

[0828] HPLC conditions above: Equipment: Agilent 1200; Column: Waters XBridge C18 4.6 x 50 mm, 3.5 μm; Flow rate: 2.0 mL / min; Gradient eluent: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00 min-1.50 min-2.50 min-2.52 min-3.00 min; Temperature: 40 °C; Phase: A: acetonitrile, B: H2O (0.05% TFA); Wavelength: 214 nm / 254 nm.

[0829] Preparation of Linker-Support 3

[0830]

[0831] opSu is a mixture of

[0832] Preparation of compound 13 (step A)

[0833] Weigh 4.33 g of Fmoc-Gly-Gly-OH and 6.84 g of Pb(OAc)4, and add to a 500 mL single neck round bottom flask. Add anhydrous THF / toluene (120 mL / 40 mL) under nitrogen atmosphere, and stir to dissolve. Then add 1.16 mL of pyridine to the reaction system. Heat the reaction system to 80 °C under nitrogen atmosphere, and reflux for 5 hours. Take sample, and monitor the reaction by HPLC detection.

[0834] Cool the reaction system to room temperature, filter, and wash the filter cake with EA for 3 times. Combine the filtrate and concentrate to dryness. Perform column chromatography (PE:EA = 100:0-50:100) to obtain about 2000 mg of the target product as a white solid with a yield of 44%.

[0835] Preparation of compound 15 (step B)

[0836] Weigh 200 mg of compound 13, and add to a 100 mL single neck round bottom flask. Then add 15 mL of THF, and stir to dissolve. Subsequently add compound 14 (312 mg, 3.0 eq) and TsOH-H2O (15 mg, 0.15 eq) to the reaction system. React the reaction system at room temperature overnight. Take sample, and monitor the reaction by TLC (PE / EA = 1:1) detection. The starting material is substantially consumed, and a new spot is detected.

[0837] Add saturated sodium bicarbonate solution to quench the reaction. Extract with EA for 3 times. Combine the organic phase and wash with brine, dry over anhydrous magnesium sulfate, and concentrate. Purify the crude product by column chromatography (PE:EA = 5:1-1:1) to obtain about 80 mg of the target product as a colorless oil with a yield of 29%. MS m / z: [M+H] = 501.1 +

[0838] Preparation of compound 16 (step C)

[0839] Weigh 200 mg of compound 15, and add to a 100 mL single neck round bottom flask. Then add 10 mL of EtOH and 5 mL of EA to completely dissolve. Then add 40 mg of palladium on carbon to the reaction system under nitrogen atmosphere, and the reaction system is replaced with hydrogen gas for 3 times. Keep the reaction system under hydrogen atmosphere, and stir at room temperature for 0.5 hours. Take sample, and monitor the reaction by TLC (DCM / MeOH = 10:1) detection. The starting material is substantially consumed, and a new spot is detected. ​

[0840] The reaction system was filtered, and the filter cake was washed with EA for 3 times. The filtrate was combined and concentrated to dryness to give 200 mg product as a white solid in 100% yield. The product was used directly for the next reaction without purification. MS m / z: [M-H] - = 409.4.

[0841] Preparation of compound 21 (step D)

[0842] Step D-1

[0843] Weigh 2.0 g of dichloro resin and put it into a polypeptide synthesis tube. Add DCM (10 mL) and swell at room temperature for 30 minutes. Remove the solvent by vacuum suction. Wash the resin with DCM for two times, each time with 7 mL of DCM for 1 minute. Remove the solvent by vacuum suction. Then weigh compound 16 (200 mg) and add it into a 50 mL centrifuge tube. Add DCM (about 10 mL). Dissolve the solid by shaking. Add it into the above resin. Stir to immerse all the resin in the solution (if there is resin attached to the tube wall, use a small amount of DCM to wash the tube wall). Stir for 4-5 hours. After the reaction is completed, add an appropriate amount of methanol. Stir for 30 minutes. Remove the solvent by vacuum suction. Wash the resin with DMF for one time, methanol for one time, DMF for one time, methanol for one time, and DMF for two times, each time with 10 mL of solvent for 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin test. The resin is colorless and transparent, and the solution is slightly yellow, indicating that it meets the requirements for entering the next coupling reaction.

[0844] Step D-2

[0845] Perform deprotection twice by adding 10 mL of ready-made 20% piperidine / DMF solution for each reaction for 10 minutes. After the reaction is completed, remove the solution by vacuum suction. Wash the resin with DMF for two times, methanol for one time, DMF for one time, methanol for one time, and DMF for two times, each time with 10 mL of solvent for 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin test. The resin and the solution are both dark blue.

[0846] Add 563 mg of Fmoc-Phe-OH, 197 mg of HOBt into a 50 mL centrifuge tube. Then add about 7 mL of DMF. Dissolve the solid by shaking. Then add 0.24 mL of DIC. Activate for 10-30 minutes to get the activated reaction solution.

[0847] Add 3 molar equivalents of the activated reaction solution to the resin. Stir to completely immerse the resin in the solution (if there is resin stuck to the wall of the tube, use a small amount of DCM to wash the wall). Stir for 2-3 hours. After the reaction is complete, remove the solvent by vacuum suction. Wash the resin with DMF twice, methanol once, DMF once, methanol once, and DMF twice, 10 mL each time for 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin test. The resin is colorless and transparent, and the solution is slightly yellow, indicating that it meets the requirements for entering the next coupling reaction.

[0848] Step D-3

[0849] Perform two deprotections by adding 10 mL of ready-made 20% piperidine / DMF solution for 10 minutes each time. After the reaction is complete, remove the solution by vacuum suction. Wash the resin with DMF twice, methanol once, DMF once, methanol once, and DMF twice, 10 mL each time for 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin test. The resin and the solution are both dark blue.

[0850] Add 531 mg of Fmoc-GG-OH, 197 mg of HOBt to a 50 mL centrifuge tube. Then add about 10 mL of DMF. Dissolve the solids by shaking. Then add 0.24 mL of DIC. Activate for 10-30 minutes to get the activated reaction solution.

[0851] Add 3 molar equivalents of the activated reaction solution to the resin. Stir to completely immerse the resin in the solution (if there is resin stuck to the wall of the tube, use a small amount of DCM to wash the wall). Stir for 2-3 hours. After the reaction is complete, remove the reaction solution by vacuum suction. Wash the resin with DMF twice, methanol once, DMF once, methanol once, and DMF twice, 10 mL each time for 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin test. The resin is colorless and transparent, and the solution is slightly yellow, indicating that it meets the requirements for entering the next coupling reaction.

[0852] Step D-4

[0853] The deprotection was performed by adding 10 mL of ready-made 20% piperidine / DMF solution for 10 minutes per reaction. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed with DMF twice, methanol once, DMF once, methanol once, and DMF twice, each time with a volume of 10 mL and a time of 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin test. The resin and solution were both dark blue. Then, 462 mg of MC-OSu was placed in a 50 mL centrifuge tube, and about 10 mL of DMF was added. The solid was dissolved by shaking. Then, 0.24 mL of DIEA was added to the resin. Agitation was performed to fully immerse the resin in the solution (if there was resin attached to the tube wall, a small amount of DCM was used to wash the tube wall). The agitation was performed for 2-3 hours. After the reaction was complete, the reaction solution was removed by vacuum suction. The resin was washed with DMF twice, methanol once, DMF once, methanol once, and DMF twice, each time with a volume of 10 mL and a time of 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin test. The resin was colorless and transparent, and the solution was slightly yellow, indicating that it met the requirements for entering the next coupling reaction.

[0854] Step D-5

[0855] The resin was washed with 10 mL of methanol twice. Then the solvent was completely removed by vacuum suction. The resin was poured out and weighed. The cleavage buffer was prepared in a 250 mL conical flask, with a ratio of TFE / DCM of 80% / 20%, and a volume of 7-8 times the weight of the peptide resin. The cleavage buffer was added to the peptide resin and shaken. The resin was fully immersed in the cleavage buffer and cleaved at room temperature for 2-3 hours. Then a simple filter made from a syringe was used to filter out the cleavage buffer, 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 cleavage buffer, shaken and left to stand for 20-30 minutes. The above system was centrifuged in a centrifuge 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, ultrasonically washed once, centrifuged at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was placed in a centrifuge tube and air-dried overnight, and then subjected to preparative purification to obtain 125 mg of product as a white solid with a yield of 40%. MS m / z: [M-H] - = 641.5.

[0856] Preparation of compound 22 (step E)

[0857] Take 150 mg of starting material compound 21 and 55 mg of TSTU, add to a 10 mL single neck round bottom flask, add anhydrous DMF (3 mL) under nitrogen atmosphere and stir for 20 minutes, then add 18 mg of compound 12-1 and 20 μL of DIEA to the reaction system in sequence. Stir at room temperature under nitrogen atmosphere for 2-8 hours. Take samples and monitor the reaction by HPLC. The starting material peak disappears completely and a new peak is detected.

[0858] The reaction system is subjected to preparation purification, the target product is collected, and freeze-drying is performed to obtain about 22 mg of product in the form of a light yellow solid. MS m / z: [M+H] + = 1081.0.

[0859] Preparation of linker-support 3 (step F)

[0860] Take compound 22 (30 mg) and add to a 10 mL single neck round bottom flask, add pure water (2 mL). Stir to dissolve. Add a DMF solution (2 mL) containing linker-support intermediate 1 (19.5 mg) to the reaction system and stir. After the reaction is carried out overnight, monitor the reaction using HPLC until all starting materials are converted to intermediates. Directly add an appropriate amount of Tris base solution or other solution to promote the ring-opening reaction to the reaction mixture, and continue to react at 0-40°C for 0.2-20 hours. Monitor the reaction by HPLC until all intermediates are consumed, and then quench with an acetic acid solution.

[0861] The reaction system is subjected to preparation purification, the target product is collected, and freeze-drying is performed to obtain about 25 mg of linker-support 3 in the form of a light yellow solid. MS m / z: [(M+3H) / 3] + = 1194.4.

[0862] Preparation of linker-support 4

[0863] The following linker-support 4 can be prepared using a similar synthetic route and reagents as linker-support 3. The structure of linker-support 4 is as follows:

[0864]

[0865] opSu is a mixture of

[0866] Example 2.3 Preparation of linker-support 5

[0867]

[0868] Step 1: Preparation of intermediate compound b

[0869] ​Step 1.1 Preparation of NH2-Asp(OtBu)-Rink amide resin

[0870] Add 2400 mL of deprotection reagent to completely remove Fmoc, then wash several times with DMF and DCM at room temperature. The resin shows blue color in the subsequent ninhydrin test.

[0871] Weigh 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT, and dissolve in 2000 mL of DMF and 80 mL of DIC solution. After placing in an ice bath at -10°C for 0.5 hours, slowly add it to the reaction kettle containing the resin, and stir at room temperature for 2-5 hours under nitrogen protection, then filter. Wash the resin with DMF and DCM in turn, and show no color or light yellow in the subsequent ninhydrin test.

[0872] Add 2400 mL of deprotection reagent to completely remove Fmoc, then wash several times with DMF and DCM at room temperature. The resin shows blue color in the subsequent ninhydrin test.

[0873] Step 1.2 Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin

[0874] Weigh 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT, and dissolve in 2000 mL of DMF and 80.0 mL of DIC solution. After placing in an ice bath at -10°C for 0.5 hours, slowly add it to the reaction kettle containing the resin, and stir at room temperature for 2-4 hours under nitrogen protection, then filter. Wash the resin with DMF and DCM in turn, and show no color or light yellow in the subsequent ninhydrin test.

[0875] Add 2400 mL of deprotection reagent to completely remove Fmoc, then wash several times with DMF and DCM at room temperature. The resin shows blue color in the subsequent ninhydrin test.

[0876] Step 1.3 Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0877] Weigh 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT, and dissolve in 2000 mL of DMF and 80 mL of DIC solution. After placing in an ice bath at -10°C for 0.5 hours, slowly add it to the reaction kettle containing the resin, and stir at room temperature for 2-4 hours under nitrogen protection, then filter. Wash the resin with DMF and DCM in turn, and show no color or light yellow in the subsequent ninhydrin test.

[0878] Add 2400 mL of de-protection reagent to completely remove Fmoc, then wash with DMF and DCM several times at room temperature. The resin shows blue color in the subsequent ninhydrin test.

[0879] Step 1.4 Preparation of 4Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0880] Weigh 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT, and dissolve in 2000 mL of DMF and 80.0 mL of DIC solution. After placing in an ice bath at -10°C for 0.5 hours, it is slowly added to the reactor containing the resin, and the reaction is stirred at room temperature for 2-4 hours under nitrogen protection, then filtered. The resin is washed with DMF and DCM in turn, and shows no color or light yellow in the subsequent ninhydrin test.

[0881] Add 2400 mL of de-protection reagent to completely remove Fmoc, then wash with DMF and DCM several times at room temperature. The resin shows blue color in the subsequent ninhydrin test.

[0882] Step 1.5 Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0883] Weigh 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT, and dissolve in 2000 mL of DMF and 80.0 mL of DIC solution. After placing in an ice bath at -10°C for 0.5 hours, it is slowly added to the reactor containing the resin, and the reaction is stirred at room temperature for 2-4 hours under nitrogen protection, then filtered. The resin is washed with DMF and DCM in turn, and shows no color or light yellow in the subsequent ninhydrin test.

[0884] Step 1.6 Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0885] Add 2400 mL of de-Dde reagent, and stir the reaction at room temperature for 0.5 hours under nitrogen protection, then filter. After repeating the operation 3 times, the resin is washed with DMF and DCM in turn, and shows blue color in the subsequent ninhydrin test.

[0886] Step 1.7 Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0887] Weigh 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT, and dissolve in 2000 mL of DMF and 80.0 mL of DIC solution. After placing in an ice bath at -10°C for 0.5 hours, it is slowly added to the reaction kettle containing the resin, and the reaction is stirred at room temperature for 2-4 hours under nitrogen protection, and then filtered. The resin is washed with DMF and DCM in turn, and shows no color or light yellow in the subsequent ninhydrin test. The resin peptide is washed with anhydrous ethanol three times, filtered and waits for cleavage.

[0888] Step 1.8: Preparation of intermediate compound b

[0889] Add 10000 mL of cleavage reagent (TFA: TIS: H2O = 95:2.5:2.5) to a 10 L reactor and cool to -10±2°C. Add the dried and weighed resin. Warm the reaction to room temperature and stir for 2-3 hours under nitrogen. After that, filter the resin and wash once with 100 mL of TFA. Combine the filtrate and washing solution.

[0890] Add 40 L of pre-cooled (below -10°C) cold ether to the product solution. Stir the mixture for 10 minutes, then centrifuge the precipitate. After centrifugation, discard the supernatant, collect the precipitate, and wash with cold ether, and centrifuge the precipitate again (set the centrifugal speed to 3600 rpm each time, centrifugal time is 5 minutes, and centrifugal cavity temperature is -5°C).

[0891] Collect the precipitate as crude compound b. Purify the crude product by preparative HPLC and lyophilize to obtain pure compound b.

[0892] Step 2: Preparation of intermediate compound a

[0893]

[0894] Step 2.1 Preparation of compound 2

[0895] Add compound 1 (1 equivalent) and DMF (5 v / v) to the reaction bottle, and the mixture is stirred and dissolved under nitrogen protection. After cooling to 0-5°C in an ice bath, add DIEA (3 equivalents) dropwise, and after the dropwise addition is complete, stir the mixture at 5°C for 10 minutes. Then add benzyl bromide (1.3 equivalents) dropwise, and after the dropwise addition is complete, allow it to naturally rise to room temperature of about 20°C, and stir for 16 hours.

[0896] The reaction solution was slowly poured into ice water, MTBE was added and stirred, then the solution was allowed to stand to separate into layers. The aqueous phase was extracted with MTBE 4 times, the combined organic phase was washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, concentrated under vacuum to obtain a yellow oil of crude product, and wet column chromatography was used. Light yellow oil was obtained by elution with PE / EA = 6:1, and the yield was 100%.

[0897] Step 2. Preparation of compound 4 of compound 2.2

[0898] Under nitrogen protection, compound 2 (2.0 eq), compound 3 (1 eq) and THF (10 v / v) were added to the reaction bottle and stirred to dissolve, TsOH (0.1 eq) was weighed and added to the reaction, and the reaction was carried out at 20-22°C for 4 hours. The reaction solution was slowly poured into ice water, extracted with EA 3 times, the combined organic phase was washed with saturated sodium bicarbonate aqueous solution, water and saturated brine in turn, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under vacuum to obtain a crude product. The silica gel sample was mixed, the product was collected by column chromatography with PE / EA = 1:1 elution, and concentrated to obtain a white solid with a yield of 40%.

[0899] Step 2. Preparation of compound 7 of compound 2.3

[0900] Under nitrogen protection, compound 4 and DMAc (10 v / v) were added to the reaction bottle and stirred to dissolve. The reaction was cooled to 14-18°C, DBU (0.5 eq) was added dropwise, the reaction was stirred at this temperature for 1.5 hours, and the raw material reaction was monitored by TLC. The reaction was cooled to 0-5°C, PPTS (0.5 eq), EDCI (1 eq), HOBT (1 eq) and compound 6 (0.85 eq) were added, and the reaction was carried out at 0-10°C for 3-4 hours, and the reaction was monitored by LCMS.

[0901] The reaction solution was added to ice water, 2-methyltetrahydrofuran was added to extract once, and the aqueous phase was extracted with 2-methyltetrahydrofuran twice. The organic phase was combined, washed with 0.5M 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 with a yield of 78%.

[0902] Step 2. Preparation of compound 10 of compound 2.4

[0903] To a reaction flask was added intermediate 7 and DMAc (10 v / v) under nitrogen protection and stirred to dissolve. The reaction was cooled to 14-18 °C, DBU (0.5 eq) was added dropwise, the reaction was stirred at this temperature for 1.5 h, the reaction was monitored to completion by TLC. The reaction was cooled to 0-5 °C, PPTS (0.5 eq), EDCI (1 eq), HOBT (1 eq) and compound 9 (0.85 eq) were added and the reaction was stirred at 0-10 °C for 3-4 h and the reaction was monitored by LCMS.

[0904] The reaction solution was added to ice water, extracted once with 2-methyltetrahydrofuran, the aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined, washed with 0.5 M hydrochloric acid, saturated aqueous NaHC03, 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 in vacuo to obtain a white solid with a yield of 50%.

[0905] Step 2.5: Preparation of compound a

[0906] Intermediate 10 was dissolved in DCM (15 v / v) under nitrogen protection, DBU (0.5 eq) was added dropwise at 20 °C and the reaction was stirred at 18-22 °C for 5 h. The reaction was monitored to completion by LCMS. The reaction solution was diluted with DCM and purified by wet column and the product was collected by elution with DCM:MeOH to obtain a white solid with a yield of 82%.

[0907] Step 3: Preparation of intermediate compound c

[0908]

[0909] Compound b (400 mg, 0.245 mmol) and compound a (377 mg, 0.539 mmol) were dissolved in DMF (6 mL), then DIPEA (159 mg, 1.23 mmol) and HATU (233 mg, 0.613 mmol) were added to the reaction solution and the reaction was stirred at room temperature for 2 h. After compound b disappeared, the product was purified by preparative HPLC, the preparation solution was lyophilized to obtain 380 mg of product with a yield of 52%. C 142 H 207 O 49 N 21 [(M+3H) / 3] + Calculated for C64H73N10O14S: 997.8, found: 875.9 (fragment mass).

[0910] Step 4: Preparation of intermediate compound d

[0911]

[0912] Compound c (380 mg, 0.245 mmol) was dissolved in pure water (80 mL) and palladium hydroxide (38 mg) was added. The system was purged with hydrogen gas three times and the reaction was stirred at room temperature for 1.5 hours. The reaction progress was monitored during the time and the reaction was stopped as soon as the starting material disappeared to prevent the increase of de-Fmoc product. The reaction solution was filtered and purified by preparative HPLC to obtain 270 mg of product with a yield of 76%. C 128 H 195 O 49 N 21 [(M+3H) / 3] + Calculated: 937.8, found: 875.9 (fragment mass).

[0913] Step 5: Preparation of intermediate compound e

[0914]

[0915] Compound 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 2-16 hours. After the reaction was completed by HPLC monitoring, the reaction mixture was directly purified by preparative HPLC, the eluate was collected and lyophilized to obtain 235 mg of product with a yield of 66%. C 174 H 229 O 55 Cl2F2N 27 [(M+3H) / 3] + Calculated: 1229.2, found: 1229.3.

[0916] Step 6: Preparation of linker-support 5

[0917]

[0918] Compound e (210 mg, 0.057 mmol) was dissolved in DMF (5 mL) and then diethylamine (0.5 mL) was added, the reaction was reacted at room temperature for 15 minutes and the reaction end point was monitored by HPLC. After the reaction was completed, it was adjusted to neutral with 10% TFA aqueous solution under ice bath, and the reaction was purified by preparative HPLC and lyophilized to obtain 145 mg of product with a yield of 73%. C 159 H 219 O 53 Cl2F2N 27 [(M+3H) / 3]+ Calculated value: 1155.2, measured value: 1155.3.

[0919] Example 3: Preparation of Targeted Molecule-Drug Conjugate

[0920] 3.1 Preparation of DS1062a analogues and Trodelvy

[0921] DS1062a analogues (DS1062a and DS-1062) * Prepared according to the method described in patent US20160297890A, or prepared by WuXi Biologics.

[0922] Trodelvy is commercially available.

[0923] 3.2 Preparation of ADC-1

[0924] 3.2.1 Processing of GQhRS7

[0925] Treat GQhRS7 via ultrafiltration, dialysis, or desalting column. Replace the storage solution with ligase buffer.

[0926] 3.2.2 Enzyme-catalyzed coupling of ADC-1

[0927] ADC-1 was prepared by coupling GQhRS7 with linker-load 1 under the catalysis of wild-type sorting enzyme A or a mutant ligase optimized based thereon. The modified antibody and linker-load were thoroughly mixed in a ligase buffer at a molar ratio of 1:1 to 1:100 and added to a solid-phase coupling system. The solid-phase coupling system contained a ligase immobilized on the matrix of the solid-phase coupling system. The immobilized ligase catalyzed the coupling reaction between antibody GQhRS7 and linker-load 1. The coupling reaction was carried out at 4°C–40°C for 0.5 h–20 h. After the reaction was complete, the reaction mixture was ultrafiltered or dialyzed to remove unreacted intermediates, yielding ADC-1. ADC-1 was stored at 4°C or -80°C in a buffer containing 20 mM citric acid, 200 mM NaCl, and pH 5.0.

[0928] 3.2.3 HIC-HPLC Detection and Analysis of ADC-1

[0929] The DAR (drug-antibody ratio) distribution of ADC-1 was analyzed by HIC-HPLC. The antibody GQhRS7, which does not contain cytotoxicity, was less than 5%; the conjugate product mainly contained ADC-1, with a DAR of 3.5.

[0930] 3.2.4 SEC-HPLC Detection and Analysis of ADC-1

[0931] The extent of high molecular weight aggregation of ADC-1 was analyzed by SEC-HPLC. The results showed that no high molecular weight polymer was detected in ADC-1, indicating that the coupling reaction condition was mild and would not cause damage to the antibody structure.

[0932] 3.3 The linker-ligand intermediates were coupled to the antibody in a site-specific manner by ligase, respectively, to form ADC. The method of coupling reaction can refer to WO2015165413A1. The obtained ADCs are listed in the following table:

[0933] ADC name Linker-payload Antibody ADC-1 Linker-payload 1 GQhRS7 ADC-2 Linker-payload 3 GQhRS7 ADC-3 Linker-payload 4 GQhRS7 ADC-4 Linker-payload 5 GQhRS7 ADC-5 Linker-payload 5 GQAb13 ADC-6 Linker-payload 5 GQAb16 ADC-7 Linker-payload 2 GQhRS7

[0934] 3.4 Binding activity

[0935] Human Trop 2 ECD at a concentration of 0.5 pg / mL was coated on a 96-well plate and incubated at 4°C overnight. Then the plate was blocked with 3% BSA-PBST for 1 hour at room temperature. After washing with PBST (0.05% Tween), a series of different concentrations of test samples (including ADC-2 (i.e. ADC2), Trodelvy and GQhRS7) were added to the 96-well plate, respectively, and then incubated at room temperature for 60 minutes. After incubation, goat anti-human FC secondary antibody (HRP) (Sinobiological, SSA001) was added at a ratio of 1:100000 and incubated at room temperature for another 60 minutes. After washing, the plate was treated with TMB solution (Sigma, T0440) as HRP substrate and the reaction was terminated with 1M H2SO4. The absorbance of each well was detected at a wavelength of 450 nm.

[0936] The results of this analysis are shown in Figure 1.2 The results show that ADC-2 has similar binding affinity to GQhRS7 and Trodelvy.

[0937] 3.5 Internalization activity

[0938] NCI-N87 with good viability were collected after trypsinization and suspended in cold FACS buffer (DPBS + 2% FBS). Cells were incubated with test drug solution at a final concentration of 50 pg / mL on ice for one hour. The antibody-cell mixture was washed twice with cold FACS buffer to remove excess antibody. Antibody-bound cells were fluorescently labeled by mixing with 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 again twice. Cells were incubated at 37°C for 10 minutes, 30 minutes, 60 minutes, 90 minutes, 150 minutes, and 210 minutes to allow internalization of the test drug. Cells were suspended by quenching buffer (150 mM NaCl + 100 mM glycine, pH = 2.0 to 2.5) to dissociate antibodies bound on the cell surface. After acidification, cells were washed twice with cold FACS buffer and analyzed by flow cytometry in the APC channel.

[0939] MFI data were substituted into the following formula and analyzed by one-phase exponential association function in Prism 8. Internalization amount: A = MFI(I) - MFI(MIN). Internalization ratio: R = [MFI(I) - MFI(MIN)] / [MFI(MAX) - MFI(Blank)] x 100%

[0940] As shown in Figure 1.3 ADC2 showed comparable internalization activity on NCI-N87 as DS1062, Trodelvy, and GQhRS7.

[0941] Effect Example 1.1 ADC-1 bystander killing effect on HepG2 in BxPC-3 / HepG2 co-culture test

[0942] Effect Example 1.1 ADC-1 bystander killing effect on HepG2 in BxPC-3 / HepG2 co-culture test

[0943] The cell concentration of Trop2-positive cells BxPC-3 and Trop2-negative cells HepG2 was adjusted to 1 x 10 6Cells were seeded at a density of 100 μL / mL in 6-well plates (BxPC-3:HepG2 = 4:1 cell volume) and supplemented with 2.8 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium. Cells were incubated overnight at 37°C with 5% CO2. 3 mL of 20 nM ADC-1 and DS1062a were added to the overnight cultured cells (final drug concentration 10 nM per well). A negative control group was set up: 3 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium was added to each well. After treatment, cells were transferred to an incubator and incubated for 96 hours. After incubation, cells were digested, washed once with 1×PBS, and then transferred to flow cytometry tubes and centrifuged at 2000 rpm for 3 minutes. The supernatant was discarded, and cell count and viability were assessed. A certain amount of cells was washed with 1×PBS, centrifuged, and the supernatant was discarded. 200 μL of 100 nM anti-human Trop2 antibody was added, and the cells were mixed and incubated at 4°C for 30 minutes. The cells were then washed with 1×PBS, centrifuged, and the supernatant was discarded. 200 μL of 5 μg / mL human IgG Fc antibody was added, and the cells were mixed and incubated at 4°C for another 30 minutes. Finally, the cells were washed with 1×PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in PBS and analyzed by flow cytometry using FlowJo software. The results are shown in Table 4.

[0944] Table 4. Bystander lethality test results for ADC-1 and DS1062a.

[0945]

[0946] Effect Example 1.2 Bystander killing effect of ADC-2 on HepG2 in BxPC-3 / HepG2 co-culture experiment

[0947] The cell concentrations of Trop2-positive BxPC-3 cells and Trop2-negative HepG2 cells were adjusted to 1×10⁻⁶. 6 Cells were seeded at 300 μL per well (BxPC-3:HepG2 = 2:1) into six-well plates, and supplemented with 2.7 mL of 45% RPMI-1640 + 45% DMEM + 10% FBS medium. Cells were incubated overnight at 37°C in a 5% CO2 incubator. 3 mL of 20 nM ADC-2 and DS1062 were added to the overnight cultured cells (final drug concentration 10 nM per well). The bystander killing effects of ADC-2 and DS1062a were evaluated using the same method as described above. The results are shown in Table 5.

[0948] Table 5. Bystander lethality test results for ADC-2 and DS1062a

[0949]

[0950] Example 1.3 Effect of ADC-6 on bystander killing of HepG2 in BxPC-3 / HepG2 co-culture test

[0951] The bystander killing effect of control (Datopotamab), DS1062a, ADC-1, ADC-2, ADC-4 and ADC-6 was evaluated using the same method as Example 1.2. The results are shown in Table 6.

[0952] Table 6 Results of bystander killing test of ADC-6

[0953]

[0954] Conclusion

[0955] The results of Example 1.1 show that both ADC-1 and DS1062a analogues have bystander killing effect, and there is no significant difference in efficacy.

[0956] The results of Example 1.2 show that the bystander killing effect of ADC-2 is better than that of DS1062a.

[0957] The results of Example 1.3 show that: ADC-1, ADC-2, ADC-4, ADC-6 and DS1062a analogues all have bystander killing effect. The bystander killing effect of ADC-2 and ADC-6 is better than that of DS1062a. The bystander killing effect of ADC-6 is significantly better than that of ADC-4.

[0958] Example 2 Effect of TROP2-targeting conjugates on cell proliferation

[0959] Example 2.1 Inhibition of tumor cell proliferation by ADC-7 and ADC-1

[0960] Example 2.1.1 Inhibition of human pharyngeal squamous cell carcinoma FaDu proliferation by ADC-7

[0961] Two thousand human pharyngeal squamous cell carcinoma FaDu with high TROP2 expression were inoculated into 96-well plates, 100 μL of culture medium per well, and incubated in a cell incubator at 37°C and 5% CO2overnight. 100 μL of ADC-7 and DS1062a of different concentrations (200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, 0.000512 nM, 0.0001024 nM) were added to each well (3 replicate wells for each concentration). A positive control group was set up: 100 μL of puromycin at a concentration of 10 μg / mL was added to each well. A negative control group was set up: 100 μL of FaDu cell complete culture medium was added to each well. After the administration was completed, the cells were transferred to the incubator for incubation for 96 hours. The 96-well plate was taken out of the 37°C cell incubator and equilibrated to room temperature for 30 minutes. After the culture medium was discarded, 100 μL of DMEM and 50 μL of CellTiter Glo reagent were added to each well, the cells were shaken at 200 rpm in the dark for 15 minutes, and the luminescent signal reflecting cell viability was detected with 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.

[0962] Table 7 Inhibitory effect of ADC-7 and DS1062a on the proliferation of pharyngeal squamous cell carcinoma FaDu

[0963] Drug IC 50 (nM) ADC-7 0.4617 DS1062a 0.5508

[0964] Effect Example 2.1.2 Inhibitory effect of ADC-1 on the proliferation of human pancreatic cancer cells BxPC-3

[0965] The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human pancreatic cancer cells BxPC-3 (100 μL / well, containing 2000 cells), and the same method was used to evaluate the inhibitory effect of ADC-1. 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.

[0966] Table 8 Inhibitory effect of ADC-1 and DS1062a on the proliferation of human pancreatic cancer cells BxPC-3

[0967] Drug IC 50 (nM) ADC-1 0.4741 DS1062a 0.5835

[0968] Effect Example 2.1.3 Inhibitory effect of ADC-1 on the proliferation of human breast cancer cells MDA-MB-468

[0969] Example 2.1.4 Inhibitory effect of ADC-7 on the proliferation of human gastric cancer cells NCI-N87 Figure 4 and Table 9.

[0970] Table 9 Inhibitory effect of ADC-1 and DS1062a on the proliferation of human breast cancer cells MDA-MB-468

[0971] Drug IC 50 (nM) ADC-1 3.347 DS1062a 2.501

[0972] Example 2.1.4 Inhibitory effect of ADC-7 on the proliferation of human gastric cancer cells NCI-N87

[0973] Example 2.1.1 was replaced with human gastric cancer cells NCI-N87 (100 μL / well, containing 5000 cells), and the inhibitory effect of ADC-7 was evaluated by the same method. The inhibitory effect of the test drug on the proliferation of human gastric cancer cells NCI-N87 was as shown in Figure 5 and Table 10.

[0974] Table 10 Inhibitory effect of ADC-7 and DS1062a on the proliferation of gastric cancer cells NCI-N87

[0975] Drug IC 50 (nM) ADC-7 1.518 DS1062a 1.262

[0976] Example 2.2 Inhibitory effect of ADC-2 and ADC-3 on the proliferation of tumor cells

[0977] Example 2.2.1 Inhibitory effect of ADC-2 and ADC-3 on the proliferation of human pharyngeal squamous cell carcinoma FaDu

[0978] Example 2.1.1 was replaced with ADC-2, ADC-3 and ADC-1, and the inhibitory effect was evaluated by the same method. The inhibitory effect of the test drug on the proliferation of human pharyngeal squamous cell carcinoma FaDu was as shown in Figure 6 and Table 11.

[0979] Table 11 Inhibitory effect of ADC-2, ADC-3 and ADC-1 on the proliferation of pharyngeal squamous cell carcinoma FaDu

[0980] Drug IC 50 (nM) ADC-2 0.2376 ADC-3 0.1761 ADC-1 0.1618

[0981] Example 2.2.2 Inhibitory effect of ADC-2 and ADC-3 on the proliferation of human pancreatic cancer cells BxPC-3

[0982] The human pharyngeal squamous cell carcinoma FaDu in Example 2.2.1 was replaced by human pancreatic cancer cell BxPC-3, and the inhibitory effect of ADC-2 was evaluated by the same method. The inhibitory effect of the test drug on the proliferation of human pancreatic cancer cell BxPC-3 was evaluated by the same method as Figure 7.1 and Table 12.

[0983] Table 12 Inhibitory effect of ADC-2, ADC-3 and ADC-1 on the proliferation of human pancreatic cancer cell BxPC-3

[0984] Drug IC 50 (nM) ADC-2 0.4526 ADC-3 0.6909 ADC-1 0.8316

[0985] Effect Example 2.3 Inhibitory effect of ADC-2 and DS-1062a on the proliferation of BxPC-3, FaDu and NCI-N87

[0986] Trop2-positive cancer cells BxPC-3 Figure 7.2 ), FaDu Figure 7.3 ) and NCI-N87 Figure 7.4 ) were used for cytotoxicity assay to analyze the effect of conjugates on tumor cell proliferation. The test drugs included conjugates ADC2, DS1062a and GQhRS7. Briefly, 3000 to 5000 cells were seeded in 96-well plates and the cells were allowed to attach overnight. The cells were treated with different concentrations of the specified drugs for 168 hours. Cell viability was detected by luminescent cell viability assay, and the percentage of cell viability was calculated.

[0987] In Trop2-positive BxPC-3, FaDu and NCI-N87, ADC2 showed stronger cytotoxicity than DS1062a. The IC 50 value of ADC2 was lower than that of DS1062a (see table below).

[0988]

[0989] Conclusion

[0990] The results of Effect Example 2.1.1 showed that both ADC-7 and DS1062a could inhibit the proliferation of FaDu cells, and the inhibitory effect of ADC-7 was slightly better.

[0991] The results of Effect Example 2.1.2 showed that both ADC-1 and DS1062a could inhibit the proliferation of BxPC-3 cells.

[0992] The results of Effect Example 2.1.3 showed that both ADC-1 and DS1062a could inhibit the proliferation of MDA-MB-468 cells.

[0993] The results of Effect Example 2.1.4 show that both ADC-7 and DS1062a can inhibit the proliferation of NCI-N87 cells.

[0994] The results of Effect Example 2.2.1 show that ADC-2, ADC-3 and ADC-1 can inhibit the proliferation of FaDu cells.

[0995] The results of Effect Example 2.2.2 show that ADC-2, ADC-3 and ADC-1 can inhibit the proliferation of BxPC-3 cells, and the inhibitory effect of ADC-2 is slightly better than that of ADC-3 and ADC-1.

[0996] The results of Effect Example 2.3 show that ADC-2 can inhibit the proliferation of BxPC-3 cells, FaDu cells and NCI-N87 cells, and the inhibitory effect of ADC-2 is better than that of DS1062a.

[0997] Effect Example 3 in vivo pharmacodynamic evaluation test

[0998] Effect Example 3.1 in vivo pharmacodynamic evaluation of ADC-1

[0999] Effect Example 3.1.1 in vivo pharmacodynamic evaluation of ADC-1 on BxPC-3 cells

[1000] I. Collect BxPC-3 in logarithmic growth phase, and adjust the cell density to 10 x 10 6 cells / mL with Matrigel buffer (PBS:Matrigel = 1:1). Inject 0.2 mL of prepared BxPC-3 cell suspension subcutaneously into the right scapular of 6-8-week-old SPF female BALB / c nude mice.

[1001] II. Measure the tumor diameter with a caliper, and calculate the tumor volume according to the formula V = 0.5a x b 2 (where a is the longest diameter of the tumor, and b is the shortest diameter of the tumor). After 6 days of cell inoculation, when the average tumor volume is about 151 mm 3 , randomly divide the animals into a vehicle control group, a DS1062a 3 mg / kg group and an ADC-1 3 mg / kg group, with 6 animals in each group. Intravenously administer the drugs to the animals in each group via the tail vein, and administer the same volume of vehicle to the control group. Measure the tumor volume of the animals in each group twice a week within 35 days after administration, and compare the tumor volumes of all animals on day 35 between groups. Calculate the T / C and TGI values using the tumor volume. The calculation formulae are as follows: T / C% = T RTV / C RTV x 100% (T RTV : RTV of the treatment group; C RTV: RTV) of the vehicle control group. The relative tumor volume (RTV) was calculated according to the results of tumor measurement, and the calculation formula was RTV = V t / V0, wherein V0 was the average tumor volume measured at the time of grouping (i.e. D0), V t was the average tumor volume at a certain time of measurement, and T RTV was the time interval between the start of treatment and the time of measurement. RTV The same day data was taken. The calculation of TGI (%): TGI (%) = [1- (the average tumor volume at the end of administration of a certain treatment group - the average tumor volume at the start of administration of the treatment group) / (the average tumor volume at the end of treatment of the vehicle control group - the average tumor volume at the start of treatment of the vehicle control group)] x 100%.

[1002] III. After 35 days of administration, the average tumor volumes of 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) were 648 mm 3 and 847 mm 3 , respectively. The results are shown in Figure 8 and Table 13.

[1003] Table 13 Inhibition of BxPC-3 mouse xenograft tumors by ADC-1

[1004]

[1005] Conclusion

[1006] According to the results of Effect Example 3.1.1, both the ADC-1 3 mg / kg group and the DS1062a 3 mg / kg group can significantly inhibit tumor growth compared with the control group.

[1007] Effect Example 3.1.2 In vivo efficacy evaluation of ADC-1 on NCI-N87 gastric cancer cells

[1008] In Example 3.1.1, BxPC-3 cells were replaced by NCI-N87 gastric cancer cells, and after 7 days of cell inoculation, when the average tumor volume was about 227 mm 3 , 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 by a method similar to that of Effect Example 3.1.1, and the results are shown in Figure 9 and Table 14.

[1009] Table 14 Inhibition of NCI-N87 xenograft tumor by ADC-1

[1010]

[1011] Conclusion

[1012] After 32 days of administration, the average tumor volume of 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) were 697 mm 3 and 658 mm 3 , respectively, which could significantly inhibit tumor growth.

[1013] Example 3.1.3 In vivo efficacy evaluation of ADC-1 on human breast cancer BR-05-0028

[1014] The human breast cancer BR-05-0028 model (IHC 3+) was derived from a tumor sample removed by clinical surgery. The tumor sample was inoculated into P0 generation nude mice, and the tumor tissue used in this example was P5 generation. A tumor tissue with a volume of about 30 mm 3 was inoculated subcutaneously into the right back of 6-8-week-old SPF female BALB / c nude mice. After 28 days of tumor tissue inoculation, when the average tumor volume was about 171 mm 3 , the animals were randomly divided into 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 6 animals in each group. The in vivo efficacy of ADC-1 was evaluated using a method similar to that of Example 3.1.1, and the results are shown in Figure 10 and Table 15.

[1015] Table 15 Inhibition of BR-05-0028 xenograft tumor by ADC-1

[1016]

[1017] Conclusion

[1018] 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) all significantly inhibited tumor growth.

[1019] Example 3.2 In vivo efficacy evaluation of ADC-2 and ADC-3

[1020] Example 3.2.1 In vivo efficacy evaluation of ADC-2 and ADC-3 on BxPC-3 cells

[1021] Six days after cell inoculation, when the average tumor volume was about 151 mm 3 , 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-3 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-2 and ADC-3 was evaluated using a method similar to that of Example 3.1.1, and the results are shown in Figure 11 and Table 16.

[1022] Table 16 Inhibition of BxPC-3 mouse xenograft tumors by ADC-2 and ADC-3

[1023]

[1024] Conclusion

[1025] After 28 days of administration, the average tumor volumes of 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) were 116 mm 3 and 540 mm 3 , respectively, indicating that the ADC-2 3 mg / kg group significantly inhibited tumor growth.

[1026] Example 3.2.2 In vivo efficacy evaluation of ADC-2 and ADC-3 on NCI-N87 gastric cancer cells

[1027] The BxPC-3 pancreatic cancer cells in Example 3.2.1 were replaced with NCI-N87 gastric cancer cells, and 8 days after cell inoculation, when the average tumor volume was about 188 mm3 When the average tumor volume was about 123mm 3 , the animals were randomly divided into the solvent control group, the DS1062a 3mg / kg group, the ADC-1 3mg / kg group, the ADC-2 3mg / kg group, the ADC-3 3mg / kg group, 6 animals in each group. The in vivo efficacy of ADC-2 was evaluated by using a method similar to that of Effect Example 3.2.1, and the results are shown in Table 17 and Table 18. Figure 12 and Table 17.

[1028] Table 17 Inhibition of NCI-N87 mouse xenograft tumor by ADC-2

[1029]

[1030] Conclusion

[1031] After 28 days of administration, the average tumor volume of the ADC-2 3mg / kg group (T / C = 14.08%, TGI = 122.25%, p < 0.001), the ADC-3 3mg / kg group (T / C = 33.43%, TGI = 94.72%, p < 0.001), the ADC-1 3mg / kg group (T / C = 46.15%, TGI = 76.62%, p < 0.001) and the DS1062a 3mg / kg group (T / C = 40.30%, TGI = 84.96%, p < 0.001) were 89mm 3 , 212mm 3 , 293mm 3 and 255mm 3 , respectively, indicating that all the tested drugs can significantly inhibit the growth of NCI-N87 tumors.

[1032] Effect Example 3.2.3 In vivo efficacy evaluation of ADC-2 and ADC-3 on human pharyngeal squamous cell carcinoma FaDu

[1033] In Example 3.2.1, BxPC-3 cells were replaced by human pharyngeal squamous cell carcinoma FaDu, and 11 days after cell inoculation, when the average tumor volume was about 123mm 3 , the animals were randomly divided into the solvent control group, the DS1062a 3mg / kg group, the ADC-1 3mg / kg group, the ADC-2 3mg / kg group, the ADC-2 3mg / kg group, 6 animals in each group. The in vivo efficacy of ADC-2 was evaluated by using a method similar to that of Effect Example 3.2.1, and the results are shown in Table 17 and Table 18. Figure 13.1 and Table 18.

[1034] Table 18 Inhibition of FaDu mouse xenograft tumor by ADC-2

[1035]

[1036] Conclusions

[1037] After 28 days of administration, the mean tumor volumes of 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) were 0 mm 3 , 4 mm 3 , 21 mm 3 , and 28 mm 3 , respectively, indicating that all the tested drugs significantly inhibited FaDu tumor growth. In addition, at the end of the experiment, 6 mice in the ADC-2 3 mg / kg group and 3 mice in the ADC-3 3 mg / kg group had complete tumor regression.

[1038] Example 3.2.4 In vivo efficacy evaluation of ADC-2 on MDA-MB-468

[1039] MDA-MB-468 tumor cells (ATCC, HTB-132) were maintained in vitro as monolayer cultures in L-15 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of air with 0% CO2. Cells in exponential growth phase were harvested and counted for tumor inoculation. For in vivo antitumor efficacy studies, 10 x 10 6 MDA-MB-468 human breast cancer cells (Trop2 positive) were suspended in 0.2 mL of PBS containing Matrigel (1:1) and inoculated subcutaneously in the right flank of BALB / c nude mice. After 24 days, when the average tumor volume reached 187 mm 3 , tumor-bearing mice were grouped and administered intravenously 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 a week with calipers. T / C and TGI values were calculated using tumor volume.

[1040] Conclusions

[1041] After 35 days of administration, the ADC-2 4.5 mg / kg group (T / C = 0.00%, TGI = 137.93%), the DS1062a 4.5 mg / kg group (T / C = 1.35%, TGI = 136.06%) and the Trodelvy 4.5 mg / kg group (T / C = 78.73%). The results are shown in Table 8 and Figure 8. Figure 13.2 The efficacy of ADC-2 is significantly better than that of Trodelvy, and slightly better than that of DS1062a.

[1042] Inhibition of MDA-MB-468 mouse xenograft tumors by ADC-2

[1043]

[1044] Efficacy Example 3.3 In vivo efficacy evaluation of ADC-5 and ADC-6

[1045] Efficacy Example 3.3.1 In vivo efficacy evaluation of ADC-5 and ADC-6 on BxPC-3 pancreatic cancer cells

[1046] Six days after cell inoculation, when the average tumor volume was about 159 mm 3 , 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 a method similar to that of Efficacy Example 3.1.1, and the results are shown in Table 19 and Figure 19. Figure 14

[1047] Table 19 Inhibition of BxPC-3 mouse xenograft tumors by ADC-5 and ADC-6

[1048]

[1049] Conclusion

[1050] After 42 days of administration, the average tumor volumes of 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) were 8 mm 3 , 27 mm 3 and 302 mm 3 , respectively, indicating that all the tested drugs significantly inhibited tumor growth; the inhibitory effects of ADC-5 and ADC-6 were much better than that of DS1062a. ​

[1051] Example 3.3.2 In vivo efficacy evaluation of ADC-5 and ADC-6 on NCI-N87 gastric cancer cells

[1052] The BxPC-3 pancreatic cancer cells in Example 3.3.1 were replaced by NCI-N87 gastric cancer cells, and after 6 days of cell inoculation, when the average tumor volume was about 196 mm 3 , 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 in a similar manner as in Example 3.3.1, and the results are shown in Table 19 and Table 20. Figure 15

[1053] Table 20 Inhibition of NCI-N87 mouse xenograft tumors by ADC-5 and ADC-6

[1054]

[1055] Conclusion

[1056] After 42 days of administration, the average tumor volumes of 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) were 133 mm 3 , 48 mm 3 , and 437 mm 3 , respectively, indicating that all the tested drugs can significantly inhibit tumor growth; the inhibitory effects of ADC-5 and ADC-6 are much better than that of DS1062a.

[1057] Example 3.3.3 In vivo efficacy evaluation of ADC-5 and ADC-6 on human pharyngeal squamous cell carcinoma FaDu

[1058] The BxPC-3 pancreatic cancer cells in Example 3.3.1 were replaced by human pharyngeal squamous cell carcinoma FaDu, and after 10 days of cell inoculation, when the average tumor volume was about 119 mm 3 , 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 in a similar manner as in Example 3.3.1, and the results are shown in Table 19 and Table 20. Figure 16 The BxPC-3 pancreatic cancer cells in Example 3.3.1 were replaced by human pharyngeal squamous cell carcinoma FaDu, and after 10 days of cell inoculation, when the average tumor volume was about 119 mm​

[1059] Table 21 Inhibition of FaDu mouse xenograft tumor by ADC-5 and ADC-6

[1060]

[1061] Conclusion

[1062] After 31 days of administration, the average tumor volumes of 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) were 217 mm 3 , 1 mm 3 , and 296 mm 3 , respectively, indicating that all the tested drugs can significantly inhibit tumor growth; the inhibitory effect of ADC-6 is much better than that of DS1062a.

[1063] Effect Example 4 Serum stability of ADC-1

[1064] An appropriate amount of Trop 2 was covalently coupled with CNBr-activated sepharose microspheres to form an immobilized antigen. After blocking, a certain proportion of plasma pre-incubated stable samples were added and incubated with shaking. The ADC-1 and DS1062a (ADC drug) in the matrix were specifically captured by the immobilized antigen to form a solid-phase antigen / antibody complex, and the unbound substances were removed by washing. After incubation, the conjugated sugar chain of the Fc region of the ADC drug antibody was removed by N-glycosidase, followed by elution and recovery of ADC with formic acid, and detection of DAR by LC-MS. The results are shown in Figure 17 .

[1065] Conclusion

[1066] The results show that the serum stability of ADC-1 is significantly better than that of DS1062a.

[1067] Effect Example 5 In vivo efficacy evaluation of the combination of ADC2 and anti-mPD-1 antibody in MC38-hTROP2 colon cancer syngeneic CDX model

[1068] Purpose of the experiment: To evaluate the in vivo anti-tumor efficacy of the combination of ADC drug ADC2 and anti-mPD-1 on mice carrying a colon cancer Trop2 high expression CDX model.

[1069] I. Collect and count the MC38-hTROP2 cells (Biocytogen) in the exponential growth phase for tumor inoculation. 0.5 x 106 The cells were subcutaneously injected into the right flank of 6-8 week-old SPF female C57BL / 6J mice.

[1070] II. Six days after inoculation, the tumor diameters were measured with a caliper, and the tumor volume was calculated according to the formula V = 0.5a x b 2 (where a is the long diameter of the tumor, and b is the short diameter of the tumor). When the average tumor volume was about 100 mm 3 -300mm 3 , the mice were randomly divided into a vehicle group, an ADC2 3 mg / kg group, a DS1062a 3 mg / kg group, an anti-mPD-1 antibody (Bio X cell, 825822J1) 1 mg / kg group, an ADC2 + anti-mPD-1 antibody combination group, and a DS1062a + anti-mPD-1 combination group. Each group included 6 mice. The day of the first administration was defined as day 0. The vehicle group mice were given the solvent of ADC2 and the solvent of anti-mPD-1 antibody at the same frequency and administration route. The tumor volume of each group of mice was measured twice a week. The experiment was ended on day 28, and the tumor growth inhibition rate (TGI) was calculated as follows: TGI (%) = [1 - (average tumor volume of the treatment group on the ending day - average tumor volume of the treatment group on the first day) / (average tumor volume of the vehicle group on the ending day - average tumor volume of the vehicle group on the first day)] x 100%.

[1071] III. Figure 18 The tumor volume changes of tumor-bearing C57BL / 6J mice treated with (1) vehicle, (2) ADC2 3 mg / kg, (3) DS1062 3 mg / kg, (4) anti-mPD-1 antibody 1 mg / kg, (5) ADC2 3 mg / kg + anti-mPD-1 antibody 1 mg / kg, and (6) DS1062 3 mg / kg + anti-mPD-1 antibody 1 mg / kg are shown. Table 22 shows that, on the ending day (day 28), the average tumor volumes of the ADC2 3 mg / kg group, the DS1062a 3 mg / kg group, the anti-mPD-1 antibody 1 mg / kg group, the ADC2 3 mg / kg + anti-mPD-1 antibody 1 mg / kg group, and the DS1062a 3 mg / kg + anti-mPD-1 antibody 1 mg / kg group were 580 mm 3 , 1415 mm 3 , 846 mm 3 , 95 mm 3 , and 934 mm 3 , respectively, and the TGIs were 68.6%, 12.8%, 50.8%, 101.0%, and 45.0%, respectively.

[1072] The results showed that ADC2 3 mg / kg and anti-mPD-1 antibody 1 mg / kg as monotherapy could inhibit the growth of tumor cells. The combination of DS1062a and anti-mPD-1 antibody inhibited tumor growth, and its inhibitory effect was better than that of DS1062a monotherapy, but similar to that of anti-mPD-1 antibody monotherapy. The combination of ADC2 and anti-mPD-1 antibody showed excellent anti-tumor efficacy, with 5 cases of complete remission (5 / 6 CR), and the anti-tumor activity was better than that of ADC2 monotherapy (1 / 6 CR) or anti-PD-1 antibody monotherapy.

[1073] Table 22 Tumor growth inhibition of the combination of ADC2 and anti-mPD-1 antibody in colon cancer syngeneic CDX model calculated based on tumor volume

[1074]

[1075] a. Mean ± standard error; measured on the last day of treatment;

[1076] b. TGI (%) = [1-(T 28 -T0) / (V 28 -V0)] x 100%. Wherein T0 is the average tumor volume of the treatment group on the first day of administration, T 28 is the average tumor volume of the treatment group on the 28th day after administration; V0 is the average tumor volume of the vehicle group on the first day of administration, V 28 is the average tumor volume of the vehicle group on the 28th day after administration.

Claims

1. A pharmaceutical combination comprising a conjugate and an anti-PD-1 antibody, wherein the conjugate has the structure of formula (III): in, Q represents hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb―P; M is hydrogen or LKa-LKb―P; in Each LKa is selected independently. opSu is or mixtures thereof; Each LKb is independently for L 2 ―L 1 ―B; Each B is either absent independently, or a combination of the following 1) and 2): 1) self-excissing of the spacer Sp1; and 2) a bond or a combination of one or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Heterocyclic group and -(CO)-; preferably, B is -NH-CH2-U- or absent or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-; U is absent, or is O, S or NH, preferably O or S; The condition is that Q and M are not both hydrogen; P is the load, and the load is related to part B or L of formula (III). 1 Partial connection; Each L 1 Independently defined as cleavable sequence 1, cleavable sequence 1 comprising an amino acid sequence that can be cleaved by an enzyme, and cleavable sequence 1 comprising 1-10 amino acids; Each L 2 Independently as a key; or C 2-20 Alkylenes, wherein one or more -CH2- structures in the alkylene group are optionally composed of -CR 3 R 4 -, -O-, -(CO)-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10 Heterocyclic group, phenylene substitution; wherein the alkylene group, heterocyclic group, and phenylene are each independently unsubstituted or substituted by at least one substituent, the substituent being selected from halogens, -C 1-10 Alkyl, -C 1-10 Halogenated alkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ; Ld2 and each Ld1 are independent bonds; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-, or a natural amino acid or an oligomeric natural amino acid with a degree of polymerization of 2-10, wherein the natural amino acid or oligomeric natural amino acid is independently unsubstituted or has -(PEG) on its side chain. j -R 11 replace; -(PEG) t -、-(PEG) i - and -(PEG) j Each of the segments is a PEG fragment, the PEG fragment containing a specified number of continuous -(O-C2H4)- structural units or continuous -(C2H4-O)- structural units, with an optional additional C at one end. 1-10 Alkylene; R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 Each is independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 cyclohexane; or R 1 and R 2 Together with the carbon atoms they are attached to, they form 3-6 membered cycloalkyl groups; or R 3 and R 4 Together with the carbon atoms they are attached to, they form 3-6 membered cycloalkyl groups; R 11 C 1-10 alkyl; m can be any integer from 1 to 3; n is any integer from 2 to 20; d is 0, or any integer from 1 to 6; Each i is an independent integer from 0 to 100, preferably 0 to 20; more preferably, each i is an independent integer from 0 to 12; more preferably 0 to 8; especially 4; Each j is an independent integer from 1 to 100, preferably 1 to 20; preferably, each j is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12; Each t is an independent integer from 1 to 100, preferably 1 to 20; more preferably, each t is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12; A is an anti-TROP2 antibody or its antigen-binding fragment, preferably modified and bound to G in formula (III). n Partially linked, and G is glycine; z is an integer from 1 to 20.

2. The drug combination according to claim 1, wherein The coupling has a structure of formula (III-a) or (III-b):

3. The pharmaceutical combination according to claim 1 or 2, wherein... The coupling has the following structure: Preferably, z is 1 to 4; more preferably 2; i, i1, i2, i3, and i4 are each independently an integer from 0 to 100, preferably from 0 to 20; more preferably, i, i1, i2, i3, and i4 are each independently an integer from 0 to 12; more preferably from 0 to 8; especially 4; Each j is an independent integer from 1 to 100, preferably 1 to 20; more preferably, each j is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12; Preferably, n is 3, L 2 -(CH2) p -(CH2)2(CO)- or -(C2H4-O) p -(CH2)2(CO)-, p is 2 to 4, L 1 The value is Gly-Gly-Phe-Gly, and B is -NH-CH2-U- or does not exist, or is -NH-CH2-U-(CR). 1 R 2 ) g -(CO)-, U does not exist or U is 0, g is 1; Each t is an independent integer from 1 to 100, preferably 1 to 20; more preferably, each t is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12; m is any integer from 1 to 3; in particular, 1 or 2.

4. The pharmaceutical combination according to any one of claims 1 to 3, wherein The loading material is a cytotoxin or a fragment thereof, optionally derivatized to react with the B portion or L portion of the compound of formula (III) as defined in claim 1. 1 Partial connection; Preferably, the cytotoxicant is selected from taxanes, maytansine alkaloids, auristatin, epromycin, cobstatin A-4 phosphate, cobstatin A-4 and its derivatives, indole-sulfonamides, such as vincristine, vinblastine, vinorelbine, vinflunine, vinblastine esters, dehydrated vincristine, salivarius toxin 10 and its analogues, leucocelein B, eribulin, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'- Benzo[azo]azolyl-coumarin (DBC), sponge lactone, lelimycin, camptothecin and its derivatives, mitoxantrone, mitoxantrone hydrazone, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carboquinone, metoprolol, urotepa, danendomycin, esperamycin, neomycin, aclarubicin, actinomycin, atrazomycin, bleomycin, actinomycin C, carabinin, erythromycin, anticancer erythromycin, erythromycin D, daunorubicin, detoxin Doxorubicin, Epirubicin, Isorubicin, Idarubicin, Methamphetamine, Mitomycin, Nopramine, Oligomycin, Pelomycin, Pofibromycin, Purulin, Ferrous Oxybenzoycin, Rodobicin, Streptomycin, Streptozotocin, Netostatin, Zorobacterium, Trichoderma, T-2 Toxin, Veracrulin A, Bacitracin A, Serpentin, Ubenimex, Diazoserine, 6-Diazo-5-oxo-L-leucine, Folic Acid, Methotrexate, Pteroxate, Trimethoprim Sa, Idatraxa, Fludarabine, 6-Mercaptopurine, Thiomipril, Thioguanine, Ancitabine, Gemcitabine, Enoxabine, Azacitidine, 6-azauridine, Carmoflu, Cytarabine, Dideoxyuridine, Deoxyfluorouridine, Fluorouracil, Calotestosterone, Drotahistosterone Propionate, Cyclothothermone, Medanone, Testrolide, Aminoglutamate, Mitotan, Tralostan, Flutamide, Nilumet, Bicalutamide, Leuprolide Acetate, Protein kinase inhibitors and Proteasome inhibitors; and / or Selected from vincristine alkaloids, colchicine, taxanes, auristatin, maytanyl alkaloids, chachiin, doxorubicin, docamycin, SN-38, candidacin analogues, delutecan, ducamycin, chachiin, sentamycin, dolasstatin, and pyrrolobenzodiazepines. Icinotecan and its derivatives; and / or Selected from auristatin, especially MMAE, MMAF, or MMAD; and / or Selected from ixotecan and its derivatives, such as DX8951f.

5. The pharmaceutical combination according to any one of claims 1 to 4, wherein The load has the structure of formula (i): in, a* is 0 or 1; The carbon atoms labeled p1* and p2* are each asymmetry centers, and the asymmetry centers are S configuration, R configuration or racemic; L 1* Selected from C 1-6 Alkylene, the C 1-6 The alkylene group is either unsubstituted or substituted with one substituent selected from halogens, -OH, and -NH2; M* is -CH2-, -NH-, or -O-; L 2* C 1-3 Alkylene; R 1* and R 2* Each is independently selected from hydrogen and C. 1-6 Alkyl, halogen and C 1-6 Alkyl group.

6. The pharmaceutical combination according to claim 5, wherein L 1* Selected from C 1-6 straight-chain alkylene, C 1-6 Branched alkylene, C 3-6 Cycloalkylene and C 3-4 Cycloalkyl-C 1-2 A straight-chain alkylene group, wherein the alkylene and cyclic alkylene groups are each independently unsubstituted or substituted by a substituent selected from halogens, -OH, and -NH2; preferably, L 1* Selected from C 1-4 Alkylene, wherein the alkylene is unsubstituted or substituted by a substituent selected from halogens, -OH, and -NH2; more preferably, L 1* Selected from -CH2-, -C2H4-, Or each of them is independently substituted by at least one substituent selected from halogens, -OH, and -NH2; most preferably, L 1* Selected from -CH2- The "#" symbol marks the position where the carbonyl group is attached.

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

8. The pharmaceutical combination according to any one of claims 5 to 7, wherein R 1* Selected from C 1-6 Alkyl, halogen; preferably, R 1* It is either methyl or Cl.

9. The pharmaceutical combination according to any one of claims 5 to 8, wherein R 2* Selected from C 1-6 Alkyl, halogen; preferably, R 2* It is F.

10. The pharmaceutical combination according to any one of claims 1 to 3, wherein the loading material is selected from... Especially selected from 11. The pharmaceutical combination according to any one of claims 1 to 3, wherein the conjugate is selected from... Each g is an independent integer from 1 to 6, preferably from 1 to 3; more preferably 1; R 1 and R 2 Each is independently selected from hydrogen, halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, C 4-10 cyclohexene alkyl; or R 1 and R 2 Together with the carbon atoms they are attached to, they form 3-6 membered cycloalkyl groups; preferably R 1 and R 2 It is hydrogen; Each t is an independent integer from 1 to 100, preferably 1 to 20; more preferably, each t is an independent integer from 1 to 12; more preferably 8 to 12; especially 8 or 12; m is any integer from 1 to 3; in particular, 1 or 2; z is an integer from 1 to 20; in particular 2 or 4, more preferably 2.

12. The pharmaceutical combination of claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (V0). H ) and light chain variable region (V L ),in The V H Include: (i)HCDR1, wherein HCDR1 comprises the amino acid sequence X1X2GMX3 (SEQ ID No:1), wherein X1 is N, T or A, X2 is Y or A, and X3 is N or Q; (ii) HCDR2, wherein HCDR2 contains the amino acid sequence WINTX4X5GX6PX7YX8X9DFKG (SEQ ID NO:2), wherein, X4 is Y, H or D, X5 is T or S, X6 is E or V, X7 is T or K, X8 is T or A, and X9 is D or E. (iii) HCDR3, wherein HCDR3 comprises X 10 The amino acid sequence of GFGSSYWYFDV (SEQ ID NO:3), where X 10 For G or S; and / or The V L Include: (i) LCDR1, wherein the LCDR1 contains the amino acid sequence of KASQDVSIAVA (SEQ ID NO:13) or KASQDVSTAVA (SEQ ID NO:14); (ii) LCDR2, wherein LCDR2 comprises the amino acid sequence SASYRYT (SEQ ID NO: 15); and (iii) LCDR3, wherein the LCDR3 contains the amino acid sequence of QQHYITPLT (SEQ ID NO:16).

13. The pharmaceutical combination according to claim 12, wherein, The V H Include: (i) HCDR1, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:4, (ii) HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:8, and (iii) HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:11; And / or the V L Include: (i) LCDR1, wherein LCDR1 contains the amino acid sequence of SEQ ID NO:13, (ii) LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and (iii) LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:16; or the V H Include: (i) HCDR1, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:5, (ii) HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:9, and (iii) HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:12; And / or the V L Include: (i) LCDR1, wherein LCDR1 contains the amino acid sequence of SEQ ID NO:14, (ii) LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and (iii) LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:16; or The V H Include: (i) HCDR1, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:4, (ii) HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:10, and (iii) HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:11; And / or the V L Include: (i) LCDR1, wherein LCDR1 contains the amino acid sequence of SEQ ID NO:13, (ii) LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and (iii) LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:16; or The V H Include: (i) HCDR1, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:7, (ii) HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:10, and (iii) HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:11; And / or the V L Include: (i) LCDR1, wherein LCDR1 contains the amino acid sequence of SEQ ID NO:13, (ii) LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and (iii) LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:16; or The V H Include: (i) HCDR1, wherein HCDR1 contains the amino acid sequence of SEQ ID NO:6, (ii) HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:8, and (iii) HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:11; And / or the V L Include: (i) LCDR1, wherein LCDR1 contains the amino acid sequence of SEQ ID NO:13, (ii) LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and (iii) LCDR3, wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO:

16.

14. The pharmaceutical combination according to claim 12 or 13, wherein the V H Contains an amino acid sequence having at least about 90% sequence identity with the amino acid sequences of SEQ ID NO:21 to 25, and / or The V L An amino acid sequence that has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:

27.

15. The pharmaceutical combination according to any one of claims 12 to 14, wherein the antibody or antigen-binding fragment comprises: a heavy chain constant domain (CH), said CH comprising 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 comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:

29.

16. The pharmaceutical combination according to any one of claims 12 to 15, wherein the antibody or antigen-binding fragment has an equilibrium dissociation constant (K0) of about 0.5 nM to about 20 nM. D Combine with TROP2.

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

35.

18. The drug combination of claim 17, wherein the antibody or the antigen-binding fragment comprises 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 the ligase donor substrate are sequentially linked; Sp2 is a spacer sequence selected from GA, GGGGS, GGGGSGGGGS, and GGGGSGGGGSGGGGS; the recognition sequence of the ligase donor substrate is LPXTGJ, wherein... X can be any single amino acid, whether natural or non-natural; J is absent or is an amino acid fragment containing 1-10 amino acids.

19. The pharmaceutical combination of claim 18, wherein the modified antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NO:30 to 33, and / or the light chain of SEQ ID NO:40 or SEQ ID NO:41; or The modified antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NO:36 to 39, and / or the light chain of SEQ ID NO:34 or SEQ ID NO:

35.

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

21. The pharmaceutical combination according to any one of claims 1 to 20, wherein 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 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.

22. The drug combination of claim 21, wherein the anti-PD-1 antibody is selected from: pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, and camrelizumab.

23. The pharmaceutical combination according to any one of claims 1 to 22, wherein the pharmaceutical combination optionally further comprises a pharmaceutically acceptable carrier.

24. A kit comprising the drug combination of any one of claims 1 to 23.

25. The kit according to claim 24, wherein the kit comprises A first packaging unit comprising the coupling element 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; and Optionally, instructions for administering the conjugate and anti-PD-1 antibody to a subject.

26. Use of the pharmaceutical combination of any one of claims 1 to 23 or the kit of claim 24 or 25 in the preparation of a medicament for treating a disease; wherein the disease is a TROP2-associated tumor.

27. The use according to claim 26, wherein the TROP2-related tumor comprises a tumor that overexpresses TROP2 or a tumor with 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 diseased subject or reducing the likelihood of disease progression, the method comprising administering a combination of drugs 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 with cancer or reducing the likelihood of cancer progression, the method comprising administering to the subject an effective amount of the conjugate of any one of claims 1 to 20, and administering to the subject an effective amount of an anti-PD-1 antibody.

31. The method of claim 30, wherein the cancer overexpresses TROP2 or the cancer 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 method according to any one of claims 30 to 32, wherein 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 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.

34. The method according to any one of claims 30 to 32, wherein the anti-PD-1 antibody is selected from: pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, and camrelizumab.

35. The method according to any one of claims 30 to 34, wherein the coupling agent is Alternatively, the coupling agent may be 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 formulation.

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 times.

39. Use of an effective amount of the conjugate of any one of claims 1 to 20 and an effective amount of the anti-PD-1 antibody in the preparation of a medicament for treating a subject suffering from cancer.

40. The use according to claim 39, wherein the cancer overexpresses TROP2 or the cancer 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 use according to any one of claims 39 to 41, wherein 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 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.

43. The use according to any one of claims 39 to 41, wherein the anti-PD-1 antibody is selected from: pembrolizumab, nivolumab, toripalimab, tislelizumab, sintilimab, and camrelizumab.

44. The use according to any one of claims 39 to 43, wherein the coupling agent is Alternatively, the coupling agent may be ADC-2.

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

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

47. The use according to any one of claims 39 to 44, wherein the conjugate and the anti-PD-1 antibody are administered at different times.

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