Antibodies, linkers, loads, conjugates, and uses thereof

Abstract

The present application relates to antibody, load and linker molecules for targeting molecule-drug conjugates, as well as the corresponding conjugates, preparation and use thereof.

Description

[0001] This application is a divisional application of Chinese patent application No. 202380014453.7, filed on July 14, 2023, entitled “Antibodies, Linkers, Loaders, Conjugates and Applications Thereof”. Technical Field

[0002] The present application relates to the field of biopharmaceuticals, and in particular, to antibodies, loads and linkers for preparing targeting molecule-drug conjugates, as well as corresponding conjugates, preparation methods and uses thereof. Background Art

[0003] TROP2 is a transmembrane protein and has been found to be overexpressed in several cancer types including endometrial cancer, prostate cancer, pancreatic cancer, colon cancer, gastric cancer, oral cancer, and glioma, making it a natural candidate for the development of targeted therapies. TROP2 acts as a regulatory factor in cell self-renewal, proliferation, and transformation. Experiments have shown that TROP2 can promote tumor growth, and when the TROP2 gene is knocked out, tumor cell proliferation is disturbed. The limited tissue expression of TROP2 will reduce the toxicity of the treatment, which is also an advantage of the therapy targeting TROP2. A variety of ADCs targeting TROP2 have been proposed, such as DS-1062, TRODELVY, and BAT8003. These therapeutic agents significantly improve the survival rate of patients with TROP2-positive cancers.

[0004] DS-1062 is an antibody conjugate developed by Daiichi Sankyo using its proprietary DXd ADC technology. It consists of a monoclonal antibody targeting the Trop2 protein linked to DXd. Data including a large number of NSCLC patients showed that DS-1062 exhibited good dose-dependent anti-cancer activity. As the dose increased, more and more NSCLC patients' tumors shrank. TRODELVY is the first FDA-approved ADC specifically for recurrent or refractory metastatic TNBC, and the first FDA-approved anti-TROP2 ADC. It consists of an antibody targeting TROP2 linked to SN-38, an active metabolite of the chemotherapeutic drug irinotecan.

[0005] However, there is still a great demand for novel ADC drugs targeting TROP2, and high-safety ADCs are still a development direction for novel drugs. Summary of the invention

[0006] In a first aspect, a compound of formula (I) is provided:

[0007]

[0008] in

[0009] W is hydrogen, LKb or -C2H4-(PEG) t -(CO)NH2;

[0010] Y is hydrogen or LKa-LKb;

[0011] Provided that W and Y are not both hydrogen;

[0012] Each LKa is independently selected from opSu is or a mixture thereof;

[0013] Each LKb is independently L 2 ―L 1 ―B;

[0014] Each B is independently a terminal group R 10 , or a combination of the following 1), 2) and 3): 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ;

[0015] R 10 is hydrogen, or a group that can leave when reacting with a group in the supported substance;

[0016] 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;

[0017] L 2 is a chemical bond; or C 2-20 Alkylene, wherein one or more -CH2- structures in the 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 wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ;

[0018] Ld2 and each Ld1 are independently a chemical bond; or selected from -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, wherein the natural amino acid or the oligomeric natural amino acid is independently unsubstituted or its side chain is substituted with -(PEG) j -R 11 replace;

[0019] -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units with an optional additional C at one end 1-10 Alkylene;

[0020] 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

[0021] R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group; or

[0022] R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group;

[0023] R 11 It is C 1-10 alkyl;

[0024] m is any integer from 1 to 3;

[0025] n is any integer from 2 to 20;

[0026] d is 0, or any integer from 1 to 6; each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12; more preferably an integer from 0 to 8; specifically 4;

[0027] Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably an integer from 8 to 12; specifically 8 or 12;

[0028] Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably, an integer from 8 to 12; specifically, 8 or 12.

[0029] In a second aspect, a compound having the structure of formula (II) is provided:

[0030]

[0031] in

[0032] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0033] M is hydrogen or LKa-LKb―P;

[0034] Provided that Q and M are not both hydrogen;

[0035] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0036] n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I);

[0037] Preferably, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or is a combination of the following 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Heterocyclylene and -(CO)-;

[0038] Preferably, Sp1 is selected from PABC, acetal, heteroacetal and a combination thereof; more preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-; wherein the -O- or the -NH- is connected to the cleavable sequence 1, and U is not present, or is O, S or NH, preferably O or S.

[0039] In a third aspect, an anti-TROP2 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and light chain variable region (V L ),in

[0040] The V H Include

[0041] (i) a HCDR1 comprising the amino acid sequence X1AGMN (SEQ ID NO: 56), wherein X1 is N or A;

[0042] (ii) a HCDR2 comprising the amino acid sequence WINTDSGEPTYTDDFKG (SEQ ID NO: 10) or WINTYTGEPTYTDDFKG (SEQ ID NO: 8); and

[0043] (iii) a HCDR3 comprising the amino acid sequence GGFGSSYWYFDV (SEQ ID NO: 11); and / or

[0044] The V L Include:

[0045] (i) LCDR1 comprising the amino acid sequence KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0046] (ii) LCDR2 comprising the amino acid sequence SASYRYT (SEQ ID NO: 15); and

[0047] (iii) LCDR3 comprising the amino acid sequence QQHYITPLT (SEQ ID NO: 16).

[0048] In a fourth aspect, a cytotoxin having the structure of formula (i) is provided:

[0049]

[0050] in

[0051] a* is 0 or 1;

[0052] The carbon atoms labeled p1* and p2* are each an asymmetric center, and the asymmetric center is in the S configuration, the R configuration, or racemic;

[0053] L 1* Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2;

[0054] M* is -CH2-, -NH- or -O-;

[0055] L 2* It is C 1-3 Alkylene;

[0056] R 1* and R 2* are each independently selected from hydrogen, C 1-6 Alkyl, halogen and C 1-6 Alkoxy.

[0057] In a fifth aspect, a conjugate having a structure of formula (III) is provided:

[0058]

[0059] in

[0060] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0061] M is hydrogen or LKa-LKb―P;

[0062] Each LKa is independently selected from

[0063] opSu is or a mixture thereof;

[0064] Each LKb is independently L 2 ―L 1 ―B;

[0065] m, n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I);

[0066] Each B is independently absent, or is a combination of 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10heterocyclylene 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)-;

[0067] Provided that Q and M are not both hydrogen;

[0068] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0069] Each L 1 is independently 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;

[0070] Each L 2 independently a chemical bond; or C 2-20 Alkylene, wherein one or more -CH2- structures in the 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 wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ;

[0071] -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units with an optional additional C at one end 1-10 Alkylene;

[0072] 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

[0073] R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group; or

[0074] R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group;

[0075] Each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12; more preferably an integer from 0 to 8; specifically 4;

[0076] Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably an integer from 8 to 12; specifically 8 or 12;

[0077] Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably an integer from 8 to 12; specifically 8 or 12;

[0078] Each g is independently an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7;

[0079] A is an anti-TROP2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is linked to G of the compound of formula (I) n part; G is glycine;

[0080] z is an integer of 1-20.

[0081] In a sixth aspect, provided is a use of an anti-TROP2 antibody or antigen-binding fragment or a pharmaceutical composition thereof in the manufacture of a medicament for preventing or treating a disease; wherein the disease is a tumor.

[0082] In a seventh aspect, a polynucleotide is provided, which encodes an antibody or an antigen-binding fragment thereof.

[0083] In an eighth aspect, a vector is provided, comprising the polynucleotide.

[0084] In the ninth aspect, a host cell is provided, comprising the polynucleotide or the vector.

[0085] In a tenth aspect, a method for producing the antibody or antigen-binding fragment thereof is provided, the method comprising:

[0086] (i) culturing host cells under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and

[0087] (ii) recovering the antibody or antigen-binding fragment thereof from the host cell or cell culture thereof.

[0088] In another aspect, a pharmaceutical composition is provided, comprising a prophylactically or therapeutically effective amount of the conjugate, and at least one pharmaceutically acceptable carrier.

[0089] In another aspect, a pharmaceutical composition is provided, comprising a prophylactically or therapeutically effective amount of an antibody or antigen-binding fragment, and at least one pharmaceutically acceptable carrier.

[0090] In another aspect, provided is use of the conjugate, the antibody or the pharmaceutical composition in the manufacture of a medicament for preventing or treating a disease; wherein the disease is a tumor.

[0091] In another aspect, provided is a method for treating a subject suffering from a disease or preventing the progression of a disease, the method comprising administering the conjugate, the antibody or the pharmaceutical composition; and the disease is a tumor.

[0092] In one embodiment, the tumor is a TROP2-associated tumor.

[0093] 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 TROP2 gene mutation. In some embodiments, the disease is selected from the group consisting of: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovial tumor, mesothelioma, Ewing's tumor (Ewing's In one embodiment, the TROP2-related tumor is selected from breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial cancer.

[0094] In another aspect, an antibody drug conjugate comprising the antibody is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1.1 Showing internalization of the antibody on MBA-MD-468, Figure 1.2 Shows binding activity on Trop2 ECD, Figure 1.3 Internalization on NCI-N87 is shown.

[0096] Figure 2 Shown are the effects of ADC-7 and DS1062a on the viability of FaDu cells.

[0097] Figure 3 Shown are the effects of ADC-1 and DS1062a on the viability of human pancreatic cancer cells BxPC-3.

[0098] Figure 4 Shown are the effects of ADC-1 and DS1062a on the viability of human breast cancer cells MDA-MB-468.

[0099] Figure 5 Shown are the effects of ADC-7 and DS1062a on the viability of gastric cancer cell line NCI-N87.

[0100] Figure 6The effects of ADC-2, ADC-3 and ADC-1 on the viability of pharyngeal squamous cell carcinoma FaDu are shown.

[0101] Figure 7.1 The effects of ADC-2, ADC-3 and ADC-1 on the proliferation of human pancreatic cancer cells BxPC-3 are shown. Figure 7.2 To show the effect of ADC-2 on the proliferation of BxPC-3, Figure 7.3 To show the effect of ADC-2 on the proliferation of FaDu, Figure 7.4 The effect of ADC-2 on the proliferation of NCI-N87 is shown.

[0102] Figure 8 The inhibitory effect of ADC-1 on BxPC-3 mouse xenograft tumors was shown.

[0103] Fig. 9 The inhibitory effect of ADC-1 on NCI-N87 mouse xenograft tumors was shown.

[0104] Fig.10 The inhibitory effect of ADC-1 on BR-05-0028 mouse xenograft tumors was shown.

[0105] Fig.11 The inhibitory effects of ADC-2 and ADC-3 on BxPC-3 mouse xenograft tumors are shown.

[0106] Fig.12 The inhibitory effects of ADC-2, ADC-3, and ADC-1 on NCI-N87 mouse xenograft tumors are shown.

[0107] Figure 13.1 The inhibitory effects of ADC-2, ADC-3, and ADC-1 on FaDu mouse xenograft tumors were shown. Figure 13.2 The inhibitory effect of ADC-2 on MDA-MB-468 mouse xenograft tumors was shown.

[0108] Fig.14 The inhibitory effects of ADC-5 and ADC-6 on BxPC-3 mouse xenograft tumors are shown.

[0109] Fig.15 The inhibitory effects of ADC-5 and ADC-6 on NCI-N87 mouse xenograft tumors are shown.

[0110] Fig.16 The inhibitory effects of ADC-5 and ADC-6 on FaDu mouse xenograft tumors are shown.

[0111] Fig.17 Serum stability results for ADC-1 are shown. DETAILED DESCRIPTION

[0112] Specific embodiments are provided below to illustrate the technical content of the present application. Those skilled in the art can easily understand other advantages and effects of the present application through the contents disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments. Those skilled in the art can make various modifications and changes without departing from the spirit of the present application.

[0113] definition

[0114] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The technology used herein refers to the technology generally understood in the art, including variations and equivalent substitutions apparent to those skilled in the art. Although it is believed that the following terms can be easily understood by those skilled in the art, elaborating the following definitions will better illustrate the application. When a trade name is present herein, it refers to the corresponding commodity or its active ingredient. All patents, published patent applications and publications cited herein are hereby incorporated by reference.

[0115] When a certain amount, concentration or other value or parameter is set forth in the form of a range, a preferred range or a preferred upper limit or a preferred lower limit, it should be understood that it is equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether the range is explicitly described. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range. For example, the statement "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 statements should also be understood in a similar manner.

[0116] Unless the context clearly dictates otherwise, singular forms such as "a", "an" and "the" include plural forms. The expression "one (kind) or more (kind)" or "at least one (kind)" may refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 (kinds) or more (kinds).

[0117] The terms "about" and "approximately" when used in conjunction with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% of the specified value, or within a wider range.

[0118] The term "stoichiometric ratio" means that various substances are mixed in a certain weight ratio. For example, in this article, the active ingredient is mixed with the filler, binder and lubricant in a specified weight ratio.

[0119] The terms "optional" or "optionally" mean that the subsequently described event may but need not occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0120] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional, unstated 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, as well as elements, steps, or ingredients that are optionally present and do not substantially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0121] As used herein, the term "antibody" is used in a broad sense, and specifically includes complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they have the desired biological activity. The antibody can belong to any subtype (e.g., IgG, IgE, IgM, IgD and IgA) or subclass, and can be derived from any suitable species. In certain embodiments, the antibody is of human or murine origin. The antibody can also be a fully humanized antibody, a humanized antibody, or a chimeric antibody prepared by a recombinant method.

[0122] Monoclonal antibody is used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for a small number of possible natural mutations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" refers to the characteristic of an antibody being derived from a population of substantially homogeneous antibodies and should not be construed as requiring some specific method to prepare the antibody.

[0123] A complete antibody or full-length antibody essentially comprises one or more antigen-binding variable regions, and one or more light chain constant regions (CL) and one or more heavy chain constant regions (CH), which may include CH1, CH2, CH3 and CH4 depending on the subtype of the antibody. The antigen-binding variable region (also known as a fragment variable region, an Fv fragment) typically comprises a light chain variable region (VL) and a heavy chain variable region (VH). The constant region may be a constant region having a native sequence (such as a constant region having a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0124] Antibody fragments may include a portion of a complete antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementary determining regions (CDRs). Fab fragments are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments with the same structure produced, for example, by recombinant expression. Fab fragments contain a light chain (comprising VL and CL) and another chain, wherein the other chain contains the variable domain (VH) of the heavy chain and the constant region domain (CH1) of the heavy chain. F(ab')2 fragments are antibody fragments obtained by digesting immunoglobulins with pepsin at pH 4.0-4.5, or fragments with the same structure produced, for example, by recombinant expression. F(ab')2 fragments essentially contain two Fab fragments, wherein each heavy chain portion contains several additional amino acids, including cysteine, which forms a disulfide bond connecting the two fragments. A Fab' fragment is a fragment that contains half of a F(ab')2 fragment (one heavy chain and one light chain). Antibody fragments may contain multiple chains joined together, for example, via disulfide bonds and / or via peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids and typically contain at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that can produce immunospecific binding to an antigen when inserted into an antibody framework (e.g., by replacing the corresponding region).

[0125] The antibodies disclosed herein can be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant techniques, phage display techniques, synthetic techniques, or other techniques known in the art. For example, genetically engineered recombinant antibodies (or antibody mimics) can be expressed using a suitable culture system (e.g., E. coli or mammalian cells). Engineering can refer, for example, to the introduction of a ligase-specific recognition sequence at the end.

[0126] Cytotoxin refers to a substance that inhibits or prevents the expression activity of a cell, a cell function, and / or causes cell destruction. The toxicity of the cytotoxin currently used in ADC is higher than that of chemotherapeutic drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, apoptosis regulation. 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, vinblastines, colchicines, and dolastatins. Drugs targeting DNA can be, for example, drugs that directly destroy DNA structure or topoisomerase inhibitors. Examples of drugs that directly destroy DNA structure include, but are not limited to, DNA double-strand breakers, DNA alkylating agents, and DNA intercalators. The DNA double-strand break agent can be, for example, an enediyne antibiotic, including but not limited to dynemicin, esperamicin, neocarzinostatin, uncialamycin, etc. The DNA alkylating agent can be, for example, a DNA dialkylating agent (i.e., a DNA crosslinking agent) or a DNA monoalkylating agent. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole benzodiazepine (IGN) dimer, duocarmycin compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (such as DX8951f, DXd-(1) and DXd-(2), the structures of which are depicted below), camptothecins and anthracyclines. Drugs targeting RNA can be, for example, drugs that inhibit splicing, and examples thereof include, but are not limited to, pladienolide. Drugs targeting kinesin-mediated protein transport can be, for example, mitotic kinesin inhibitors, including, but not limited to, kinesin spindle (KSP) inhibitors.

[0127] A spacer is a structure that is located between different structural modules and can separate each structural module in space. The definition of a spacer is not limited by whether it has a certain function or whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that acts as a spacer, and examples thereof include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, such as a sequence containing two amino acids such as GA, etc.; or, for example, GGGGS (SEQ ID NO: 42), GGGGSGGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGS (SEQ ID NO: 44), etc. A self-removal spacer is a covalent component that causes two chemical bonds to be successively cleaved after activation of the protective part in the precursor: upon stimulation, the protective part (such as a cleavable sequence) is removed, thereby generating a cascade of decomposition reactions, so that smaller molecules are released sequentially in time. Examples of self-removal spacers include, but are not limited to, PABC (p-phenylmethyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0128] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is attached to the rest of the molecule by a single bond. Alkyl groups can contain 1 to 20 carbon atoms and refer to C1-C 20 Alkyl, such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl 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 group refers to a group obtained by removing a hydrogen atom from a carbon atom having one or more free valence electrons from a corresponding monovalent group. A divalent group has two attachment sites to the rest of the molecule. For example, "alkylene" or "alkylidene" refers to a straight or branched chain saturated divalent hydrocarbon radical. Examples of alkylene include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexamethylene (-C6H 12-), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene, ethylpropylene, etc.

[0129] As used herein, when a group is combined with another group, the connection of the groups may be linear or branched, as long as a chemically stable structure is formed. The structure formed by such a combination can be connected to the rest of the molecule through any suitable atom in the structure, preferably through a specified chemical bond. For example, when two or more divalent groups selected from the following are combined together to form a combination: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 The two or more divalent groups may form a linear connection with 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)-, etc. The resulting bivalent structure can be further linked to other parts of the molecule.

[0130] As used herein, the expressions "antibody-drug conjugate" and "antibody-drug conjugate" have the same meaning.

[0131] Compound of formula (I)

[0132] In one aspect, a compound of formula (I) is provided:

[0133]

[0134] in

[0135] W is hydrogen, LKb or -C2H4-(PEG) t -(CO)NH2;

[0136] Y is hydrogen or LKa-LKb;

[0137] Provided that W and Y are not both hydrogen;

[0138] Each LKa is independently selected from

[0139] opSu is or a mixture thereof;

[0140] Each LKb is independently L 2 ―L 1 ―B;

[0141] Each B is independently a terminal group R 10 , or a combination of the following 1), 2) and 3): 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ;

[0142] R 10 is hydrogen, or a group that can leave when reacting with a group in the supported substance;

[0143] Each L 1 is independently 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;

[0144] Each L 2 independently a chemical bond; or C 2-20 Alkylene, wherein one or more -CH2- structures in the 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 wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C1-10 Alkylene-OR 9 ;

[0145] Ld2 and each Ld1 are independently a chemical bond; or selected from -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, wherein the natural amino acid or the oligomeric natural amino acid is independently unsubstituted or its side chain is substituted with -(PEG) j -R 11 replace;

[0146] -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units with an optional additional C at one end 1-10 Alkylene;

[0147] 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

[0148] R 1 and R 2 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group; or

[0149] R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group;

[0150] R 11 It is C 1-10 alkyl;

[0151] m is any integer from 1 to 3;

[0152] n is any integer from 2 to 20;

[0153] d is 0, or any integer from 1 to 6;

[0154] Each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12; more preferably an integer from 0 to 8; specifically 4;

[0155] Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably an integer from 8 to 12; specifically 8 or 12;

[0156] Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably, an integer from 8 to 12; specifically, 8 or 12.

[0157] In one embodiment, L 2 Selected from: -(CH2) p -(CH2)2(CO)-, p is 0 or an integer from 1 to 5;

[0158] b is an integer from 1 to 10.

[0159] In one embodiment, p is 0 to 3; preferably 3.

[0160] In one embodiment, L 2 Selected from: -(C2H4-O) p -(CH2)2(CO)-, p is an integer from 1 to 5; more preferably, p is 2 or 4.

[0161] In one embodiment, each of the above L 2 The carbonyl group in the structure is connected to L 1 , and the other linking site is linked to opSu.

[0162] In one embodiment, each of the above L 2 The carbonyl group is connected to L 1 , and the other attachment site is attached to the amide.

[0163] Ld2 and each Ld1 are independently a chemical bond or

[0164]

[0165] Each i is independently an integer from 0 to 100;

[0166] Each j and k is independently an integer from 1 to 100.

[0167] In one embodiment, each i is independently an integer from 0 to 20. In one embodiment, each i is independently an integer from 0 to 12.

[0168] In one embodiment, each j and k is independently an integer from 1 to 20. In one embodiment, each j and k is independently an integer from 1 to 12.

[0169] In some embodiments, each i is independently an integer from 0 to 8; specifically 4.

[0170] In some embodiments, each j is independently an integer from 8 to 12; specifically 8 or 12.

[0171] In some embodiments, each k is independently an integer from 1 to 7; specifically 1 or 3 or 5.

[0172] In one embodiment, Ld2 and each Ld1 are independently a chemical bond; or a C having an amino group and a carbonyl group at both ends. 1-20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at both ends (denoted as -(PEG) i- ), or one or more natural amino acids, the natural amino acids are independently unsubstituted or their side chains are substituted with a PEG fragment of a certain length (denoted as -(PEG) j -)replace.

[0173] In one embodiment, -(PEG) i- Contains -(O-C2H4) i -or-(C2H4-O) i -, and has an optional additional C at one end 1-10 Alkylene; -(PEG) j -Contains -(O-C2H4) j -or-(C2H4-O) j -, and has an optional additional C at one end 1-10 In one embodiment, -(PEG) i -Contains -C2H4-(O-C2H4) i -or-(C2H4-O) i -C2H4-.

[0174] It should be understood that when there are two or more Ld1, B, L 2 or L 1 When the structure is Ld1, B, L 2 or L 1 The structures of are independently selected. When there are two or more R x (x is 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.), each R xIn some embodiments, the "x" in the molecule is represented with or without additional single primes (') or multiple primes (such as ", "', "", etc.), for example, R, R 1 '、R 1 ”、R 1 ”'、R 2 '、R 2 ”、R 2 ”', etc., with or without additional single or multiple apostrophes for each R x are 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, the “i” in the molecule is represented in the form of with or without additional numbers, such as i1, i2, i3, i4, etc., wherein the numbers do not indicate any order, but are only used to distinguish “i”. And each “i” with or without additional numbers is independently selected.

[0175] In one embodiment, the cleavable sequence 1 is selected from Gly-Gly-Phe-Gly (SEQ ID NO: 45), Phe-Lys, Val-Cit, Val-Lys, Gly-Phe-Leu-Gly (SEQ ID NO: 46), Ala-Leu-Ala-Leu (SEQ ID NO: 47), Ala-Ala-Ala and combinations thereof; preferably, the cleavable sequence 1 is Gly-Gly-Phe-Gly (SEQ ID NO: 45).

[0176] In one embodiment, W is hydrogen.

[0177] In one embodiment, W is -C2H4-(PEG) t- (CO)NH2, wherein t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably an integer from 8 to 12; specifically 8 or 12.

[0178] In one embodiment, R 11 It is C 1-6 The alkyl group is preferably a methyl group.

[0179] In one embodiment, n is an integer from 2 to 5, in particular 3.

[0180] In one embodiment, d is 0, or any integer from 1 to 4; preferably 0, 1, 2 or 3.

[0181] Sulfosuccinimide is unstable under physiological conditions and is prone to reverse Michael addition, resulting in cleavage at the conjugation site. In addition, when there is another thiol compound in the system, sulfosuccinimide can also undergo thiol exchange with the other thiol compound. These reactions will cause the load to fall and cause toxic side effects. In this article, when applied to the linker, the ring-opening succinimide structure no longer undergoes reverse Michael addition or thiol exchange, and therefore the product is more stable. The method of ring-opening reaction can be found in WO2015165413A1.

[0182] Compounds containing a ring-opened succinimide moiety can be purified by semi-preparative / preparative HPLC or other suitable separation means to obtain high purity compounds and defined compositions, regardless of the efficiency of the succinimide ring-opening reaction.

[0183] The portion containing the recognition sequence for the ligase acceptor or donor substrate

[0184] In one embodiment, G of the compound of formula (I) n The moiety is a recognition sequence for a ligase acceptor substrate, which facilitates the enzymatic coupling of the compound of formula (I) with the targeting molecule under ligase catalysis. The targeting molecule is optionally modified and comprises a corresponding recognition sequence for a ligase donor substrate.

[0185] In one embodiment, the ligase is a transpeptidase. In one embodiment, the ligase is selected from the group consisting of: natural transpeptidase, non-natural transpeptidase, variants thereof and combinations thereof. Non-natural transpeptidase can be but not limited to those obtained by engineering of natural transpeptidase. In a preferred embodiment, the ligase is selected from the group consisting of: natural sortase, non-natural sortase and combinations thereof. The types of natural sortase include sortase A, sortase B, sortase C, sortase D, plant lactobacillus (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 thus used to realize the specific conjugation between different molecules or structural fragments.

[0186] In some embodiments, the ligase is a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, and Lactobacillus plantarum sortase. In these embodiments, the recognition sequence of the ligase acceptor substrate is selected from the group consisting of oligoglycine, oligoalanine, and a mixture of oligoglycine / alanine with a degree of polymerization of 3-10. In a specific embodiment, the recognition sequence of the ligase acceptor substrate is G n , wherein G is glycine (Gly), and n is an integer from 2 to 10.

[0187] In another specific embodiment, the ligase is sortase A from Staphylococcus aureus. Thus, the ligase recognition sequence can be a typical enzyme recognition sequence, such as LPXTG (SEQ ID NO: 48). In yet another specific embodiment, the recognition sequence of the ligase donor substrate is LPXTGJ (SEQ ID NO: 49), and the recognition sequence of the ligase acceptor substrate is G n , wherein X can be any single natural or unnatural amino acid; J is absent or is an optionally labeled amino acid fragment comprising 1-10 amino acids. In one embodiment, J is absent. In yet another embodiment, J is an amino acid fragment comprising 1-10 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 recognition sequence of the ligase donor substrate is LPETG (SEQ ID NO: 50). In another specific embodiment, the recognition sequence of the ligase donor substrate is LPETGG (SEQ ID NO: 51).

[0188] In one embodiment, the ligase is sortase B from Staphylococcus aureus and the corresponding donor substrate recognition sequence can be NPQTN (SEQ ID NO: 52). In another embodiment, the ligase is sortase B from Bacillusanthracis and the corresponding donor substrate recognition sequence can be NPKTG (SEQ ID NO: 53).

[0189] In yet another embodiment, the ligase is sortase A from Streptococcus pyogenes and the corresponding donor substrate recognition sequence can be LPXTGJ (SEQ ID NO: 49), 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: 54).

[0190] In another embodiment, the ligase is sortase A from Lactobacillus plantarum and the corresponding donor substrate recognition sequence can be LPQTSEQ (SEQ ID NO: 55).

[0191] The ligase recognition sequence may also be other new transpeptidase recognition sequences optimized by artificial screening.

[0192] Moiety containing reactive groups

[0193] Reactive groups attached to the load

[0194] In one embodiment, B is a terminal group R 10 , and L 1 The cleavable sequence 1 in is connected to the load. In this case, B is not present in the molecule obtained by connecting the cleavable sequence 1 to the load. In one embodiment, B is used to connect to the load. In order to connect to the load, the compound of formula (I) comprises a reactive group. In one embodiment, B in the compound of formula (I) is connected to the load by an amide bond, an ester bond or an ether bond. In one embodiment, the reactive group in B in formula (I) is independently a reactive group (such as a reactive C=O part, a reactive C=CC=O part, an amino group, an amine group, a hydroxyl group, a thiol group) for condensation reaction, nucleophilic addition or electrophilic addition or a reactive group (such as a leaving group connected to O, C, N or S atoms) for substitution reaction. In one embodiment, the reactive group in B is independently selected from carboxyl, active ester, aldehyde group, amino group, amine group, hydroxyl group and thiol group. In a specific embodiment, the reactive group in B for connecting to the load is independently selected from amino group, amine group, hydroxyl group, thiol group, carboxyl and active ester.

[0195] In one embodiment, the reactive groups in B are independently amino, amine or hydroxyl groups, which react with corresponding groups in the support (such as carboxyl, sulfonic acid, phosphoryl with free-OH end, active ester, acyl chloride or isocyanate). In another embodiment, the reactive groups in B are independently carboxyl or active ester, which react with corresponding groups in the support (such as amino, amine or hydroxyl groups).

[0196] In one embodiment, the reactive groups in B are independently amino, hydroxyl or thiol groups, which react with corresponding groups in the support (such as halogen, hydroxyl, aldehyde group). In another embodiment, the reactive groups in B are independently hydroxyl groups, which react with corresponding groups in the support (such as halogen or hydroxyl).

[0197] In one embodiment, each B is independently R 10, or a combination of the following 1), 2) and 3): 1) a self-removing spacer Sp1; 2) a chemical bond or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene and -(CO)-; and 3) terminal group R 10 .

[0198] In one embodiment, Sp1 is selected from PABC, acetal, heteroacetal and a combination thereof. In one embodiment, Sp1 is acetal, heteroacetal or PABC. In one embodiment, heteroacetal is selected from N,O-heteroacetal. In one embodiment, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is connected to the 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.

[0199] In one embodiment, B is R 10 、-NH-CH2-UR 10 、-NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 or -NH-CH2-U-(CH2) g -(CO)-R 10 .

[0200] In one embodiment, R 10 is hydrogen, hydroxyl or In one embodiment, R 10 is hydrogen. In one embodiment, R 10 Is hydroxyl or

[0201] In one embodiment, R 10 It represents the structural part that will not appear in the product molecule produced by the reaction of B and the load.

[0202] Specific examples of compounds of formula (I)

[0203] In one embodiment, W is hydrogen, each LKa is In one embodiment, Formula (I) has the structure of Formula (Ia)

[0204]

[0205] In some embodiments, each LKa is In some embodiments, Formula (I) has the structure of Formula (Ib)

[0206]

[0207] In some embodiments, Ld2 is a chemical bond and d is 0. In some embodiments, the compound of formula (Ia) is as follows:

[0208]

[0209] In some embodiments, d is 0, Ld2 is In some embodiments, the compound of formula (Ia) is as follows:

[0210]

[0211] In some embodiments, d is 1, 2 or 3, Ld2 and each Ld1 are independently selected from In some embodiments, the compound of formula (Ia) is as follows:

[0212]

[0213] In some embodiments, Ld2 is d is 0. In some embodiments, the compound of formula (Ia) is as follows:

[0214]

[0215] In some embodiments, d is 1, 2 or 3, Ld2 is and each Ld1 is independently selected from In some embodiments, the compound of formula (Ia) is as follows:

[0216]

[0217] In some embodiments, d is 1, W is hydrogen, and Ld2 is and each Ld1 is independently selected from In some embodiments, the compound of formula (Ib) is as follows:

[0218]

[0219] In some embodiments, d is 1, W is -C2H4-(PEG) t -C(O)NH2, Ld2 is a chemical bond, and each Ld1 is independently selected from In some embodiments, the compound of formula (Ib) is as follows:

[0220]

[0221] In some embodiments, n is 3, L 2 Yes - (CH2)p -(CH2)2(CO)-, p is 3, L 1 is GGFG, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, and g is 1. In some embodiments, Ia-0-1 has the following structure:

[0222]

[0223] In some embodiments, Ia-0-2 has the following structure:

[0224]

[0225] In some embodiments, Ia-0-3 has the following structure:

[0226]

[0227] In some embodiments, Ia-0-4 has the following structure:

[0228]

[0229] In some embodiments, Ia-0-5 has the following structure:

[0230]

[0231] In some embodiments, Ia-1-1 has the following structure:

[0232]

[0233] In some embodiments, Ia-1-2 has the following structure:

[0234]

[0235]

[0236] In some embodiments, Ia-1-3 has the following structure:

[0237]

[0238]

[0239] In some embodiments, Ia-1-4 has the following structure:

[0240]

[0241]

[0242] In some embodiments, n is 3, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is GGFG, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, and g is 1. In some embodiments, Ib-1 has the following structure:

[0243]

[0244]

[0245] In some embodiments, n is 3, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is GGFG, B is -NH-CH2-UR 10 or -R 10 or -NH-CH2-U-(CR 1 R 2 ) g -(CO)-R 10 , U is O, and g is 1. In some embodiments, Ib-0 has the following structure:

[0246]

[0247]

[0248] In some embodiments, i is 4, g is 1, R 11 It's methyl.

[0249] In some embodiments, i is 2, g is 1, R 11 It's methyl.

[0250] In some embodiments, Ia-0-2 is as follows (Ia-0-2-1 to Ia-0-2-3):

[0251]

[0252] In some embodiments, i is 4, j is 8, g is 1, R11 In some embodiments, Ia-1-2 is as follows (Ia-1-2-1 to Ia-1-2-3):

[0253]

[0254] In some embodiments, n is 3, i is 4, j is 12, g is 1, R 11 In some embodiments, Ia-1-2 is as follows (Ia-1-2-4 to Ia-1-2-6):

[0255]

[0256]

[0257] In some embodiments, i is 4, j is 8, g is 1, m is 1, n is 3, R 11 In some embodiments, Ib-1 is as follows (Ib-1-1 to Ib-1-3):

[0258]

[0259]

[0260] In some embodiments, i is 4, j is 12, g is 1, m is 1, n is 3, R 11 In some embodiments, Ib-1 is as follows (Ib-1-4 to Ib-1-1-6):

[0261]

[0262]

[0263] In some embodiments, i is 4, j is 8, g is 1, m is 2, n is 3, R 11 In some embodiments, Ib-1 is as follows (Ib-1-7 to Ib-1-9):

[0264]

[0265]

[0266] In some embodiments, i is 4, j is 12, g is 1, m is 2, n is 3, R 11 In some embodiments, Ib-1 is as follows (Ib-1-10 to Ib-1-12):

[0267]

[0268]

[0269] In some embodiments, i is 4, t is 8, g is 1, m is 1, n is 3, R 11 In some embodiments, Ib-0 is as follows (Ib-0-1 to Ib-0-3):

[0270]

[0271]

[0272] In some embodiments, i is 4, t is 12, g is 1, m is 1, n is 3, R 11 In some embodiments, Ib-0 is as follows (Ib-0-4 to Ib-0-6):

[0273]

[0274]

[0275] In some embodiments, i is 4, t is 8, g is 1, m is 2, n is 3, R 11 In some embodiments, Ib-0 is as follows (Ib-0-7 to Ib-0-9):

[0276]

[0277]

[0278] In some embodiments, i is 4, t is 12, g is 1, m is 2, n is 3, R 11 In some embodiments, Ib-0 is as follows (Ib-0-10 to Ib-0-12):

[0279]

[0280]

[0281] Compound of formula (I) with a load

[0282] The reactive group contained in B is covalently conjugated with a loading substance containing another reactive group to obtain a compound of formula (I) with a loading substance.

[0283] In yet another aspect, there is provided a compound having a structure of formula (II)

[0284]

[0285] in

[0286] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0287] M is hydrogen or LKa-LKb―P;

[0288] Provided that Q and M are not both hydrogen;

[0289] P is a load, which is connected to the B part or L of the compound of formula (I) 1 part;

[0290] n, d, Ld1, Ld2, t, LKa and LKb are as defined in formula (I).

[0291] As defined above, in the compounds of formula (I), each LKb is independently L 2 ―L 1 ―B; each B is independently a terminal group R 10 , or a combination of the following 1), 2) and 3): 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen, or a group that can leave when reacting with a group in the support. In one embodiment, R 10 It represents the structural part that will not appear in the product molecule produced by the reaction of B and the load.

[0292] In one embodiment, P is connected to the B moiety of the compound of formula (I) to form the compound of formula (II). As defined above, R 10 It will not appear in the B-P structure of the compound of formula (II).

[0293] It should be understood that when B in the compound of formula (I) is a terminal group R 10 When R 10 Therefore, as a result, B does not exist in the B-P structure of the compound of formula (II).

[0294] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein P is a load, the load is connected to the B part or L of the compound of formula (I) 1 part; each B is independently a terminal group R 10, or a combination of the following 1), 2) and 3): 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen or a group that can leave when reacting with a group in the supported substance; R 10 It represents the structural part that will not appear in the product molecule produced by the reaction of B and the load.

[0295] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or is a combination of the following 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 In a 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 In ―B―P, B does not exist. In one embodiment, in LKa-L 2 ―L 1 In ―B―P, B is a combination of 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 In one embodiment, in LKa-L 2 ―L 1In ―B―P, B is -NH-CH2-U- or -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 connected to the carrier via an amide bond, an ester bond or an ether bond.

[0296] As defined above, when B in the compound of formula (I) is a terminal group R 10 When B is not present in the B-P structure of the compound of formula (II). In this case, it can also be understood that L 1 The cleavable sequence 1 in the formula (II) is connected to the cargo to form a compound of formula (II), wherein B is not present in the molecule obtained by connecting the cleavable sequence 1 to the cargo. Therefore, in one embodiment, P is connected to L of the compound of formula (I). 1 Thus, in one embodiment, M is LKa-L 2 ―L 1 ―B―P, and B does not exist; and M can also be expressed as LKa-L 2 ―L 1 ―P.

[0297] Load

[0298] In this article, the load can be selected from the group consisting of: small molecule compounds, nucleic acids and analogs, tracer molecules (including fluorescent molecules, etc.), short peptides, polypeptides, peptidomimetics and proteins. In one embodiment, the load is selected from the group consisting of: small molecule compounds, nucleic acid molecules and tracer molecules. In a preferred embodiment, the load is selected from the group consisting of small molecule compounds. In a more preferred embodiment, the load is selected from the group consisting of cytotoxins and fragments thereof.

[0299] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is selected from the group consisting of: taxanes; maytansines; auristatins; epothilones; combretastatin A-4, combretastatin A-4 and its derivatives; indole-sulfonamides; vinca alkaloids, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrous vinblastine; dolastatin 10 and analogs; halichondrin B. B); and eribulin; indole-3-oxoacetamide; podophyllotoxins; 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC); discodermolide; laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, such as camptothecins and their derivatives, mitoxantrone, and mitoguazone. In a preferred embodiment, the cytotoxin is selected from the group consisting of: nitrogen mustards, such as chlorambucil, chlornaphazine, cholephosphamide, estramustine, ifosfamide, mechlorethamine, oxynitrobenzene hydrochloride, melphalan, novembichin, phenamet, phenesterine, prednimustine, trofosfamide, uracil mustard. In another preferred embodiment, the cytotoxin is selected from the group consisting of: nitrosoureas, such as carmustine, flubenzuron, formoterol, lomustine, nimustine, ramustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridines.In a preferred embodiment, the cytotoxin is selected from the group consisting of benzodopa, carboquone, meturedepa and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of danemycin, esperamicin, neocarcinostatisin and aclacinomycin. In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, antramycin, bleomycins, actinomycin C, carabicin, carminomycin and cardinophyllin, carminomycin, actinomycin D, daunorubicin, detorubicin, adriamycin, epirubicin, esorubicin, Idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rodorubicin, rufocromomycin, streptozocin, zinostatin, zorubicin. In another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecene. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, mucin A, bacillocporin A and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs.In a preferred embodiment, the cytotoxin is selected from the group consisting of dimethylfolate, methotrexate, pteropterin, trimetrexate and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, tiamiprine, thioguanine. In yet another embodiment, the cytotoxin is selected from pyrimidine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone. In another embodiment, the cytotoxin is selected from the group consisting of antiadrenal agents. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of anti-androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprorelin acetate and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.In another embodiment, the cytotoxin is selected from the group consisting of: vinca alkaloids, colchicines, taxanes, auristatins, maytansines, calicheamicin, doxorubicin, duocarmucin, SN-38, scutellaria cyclotide analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, and pyrrolobenzodiazepine (PBD). In a specific embodiment, the cytotoxin is selected from the group consisting of: vinca alkaloids, colchicines, taxanes, auristatins, and maytansines.

[0300] In a specific embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f.

[0301] In another specific embodiment, the cytotoxin is a maytansinoid, such as DM1, etc. It should be noted that when using a cytotoxin comprising a thiol moiety that can react with a maleimide moiety to form a sulfosuccinimide, such as a maytansinoid, such as DM1, the cytotoxin can be directly attached via the sulfosuccinimide. In this case, it is understood that in some embodiments, the cargo and the thiol moiety together constitute the cytotoxin, and therefore, in this case, the cargo represents the remainder of the cytotoxin molecule except the thiol moiety.

[0302] In a specific embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), etc. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0303] The support contains reactive groups that can react with reactive groups in the compound of formula (I), thereby covalently conjugating the support with the compound of formula (I). Compounds without reactive groups need to be appropriately derivatized to obtain the support.

[0304] In one embodiment, the cytotoxin is a compound of formula (i)

[0305]

[0306] in

[0307] a* is 0 or 1;

[0308] The carbon atoms labeled p1* and p2* are each an asymmetric center, and the asymmetric center is in the S configuration, the R configuration, or racemic;

[0309] L 1* Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2;

[0310] M* is -CH2-, -NH- or -O-;

[0311] L 2* It is C 1-3 Alkylene;

[0312] R 1* and R 2* are each independently selected from hydrogen, C 1-6 Alkyl, halogen and C 1-6 Alkoxy.

[0313] In a specific embodiment, the cytotoxin is a compound of formula (i')

[0314]

[0315] Wherein g is any integer from 1 to 6.

[0316] In one embodiment, g* is any integer from 1 to 3, preferably 1.

[0317] 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, each of which is independently unsubstituted or substituted with a substituent selected from halogen, -OH and -NH2. 1* Selected from C 1-4 Alkylene, said alkylene being unsubstituted or substituted by a substituent selected from halogen, -OH and -NH2. In a preferred embodiment, L 1* Selected from -CH2-, -C2H4-, Each of them is independently unsubstituted or substituted by at least one substituent selected from halogen, -OH and -NH2. In a preferred embodiment, L 1* Selected from -CH2-, Wherein "#" marks the position of the carbonyl group. In a more preferred embodiment, L 1* Selected from -CH2-, Wherein "#" marks the position of attachment to the carbonyl group. In a specific embodiment, L 1* Selected from -CH2-, wherein "#" marks the position of attachment to the carbonyl group. In a preferred embodiment, the halogen is selected from F, Cl and Br, especially F.

[0318] In one embodiment, a* is 1, M* is -CH2-, -NH-, or -O-; and L 2* In another embodiment, a* is 1, M* is -CH2-, and L 2* In one embodiment, a* is 0.

[0319] In one embodiment, the carbon atom labeled with p1* is in S configuration or racemic, preferably in S configuration. In another embodiment, the carbon atom labeled with p2* is in S configuration or racemic, preferably in S configuration.

[0320] In one embodiment, R 1* and R 2* are each independently selected from hydrogen, C 1-3 Alkyl, halogen and C 1-3 In a preferred embodiment, R 1* and R 2* Each is independently selected from CH3-, F, Cl, Br and CH3O-. In one embodiment, R 1* In another embodiment, R 2* It's F.

[0321] In one embodiment, a* is 0, L 1* Selected from -CH2-, Wherein "#" marks the position of attachment to the carbonyl group. In one embodiment, a* is 1, L 1* yes M* is O, and L 2* It is -C2H4-.

[0322] In some embodiments, a* is 0, R 1* It is Cl, R 2* is F, and L 1* Selected from -CH2-, In some embodiments, a* is 0, R 1* is CH3-, R 2* is F, and L 1* Selected from Wherein "#" marks the position of attachment to the carbonyl group.

[0323] In some embodiments, a* is 1, R 1* is CH3-, R 2* It's F, L 1* yes M is O, and L 2* It is -C2H4-.

[0324] In one embodiment, the cytotoxin is selected from the following compounds; wherein the wavy bond shows the attachment site to the compound of formula (I).

[0325]

[0326] In some embodiments, the loading material is selected from DX8951f (Compound 9), DXd-(1) (Compound 10), DXd-(2) (Compound 14), (Compound 15), (Compound 16), preferably selected from DX8951f, DXd-(1), More preferably, it is selected from DXd-(1), Most preferably selected from

[0327] Preparation of the compound of formula (I) with a load

[0328] In one embodiment, the linker unit and the loading substance are linked via the reactive groups defined above using any reaction known in the art, including but not limited to condensation reaction, nucleophilic addition, electrophilic addition, and the like.

[0329] In one embodiment, the cargo is a cytotoxin. In one embodiment, the linker-cargo intermediate (numbered LBx) is as shown in the following table.

[0330]

[0331]

[0332] *: For all linkers listed, n is 3.

[0333] Conjugates and their preparation

[0334] In addition, the compound of formula (I) with a load having a portion containing a ligase recognition sequence can be coupled to other molecules containing a ligase recognition sequence, and can thus be used, for example, to prepare a targeting molecule-drug conjugate, such as an antibody-drug conjugate. Therefore, in yet another aspect, a conjugate is provided, the conjugate comprising a compound of formula (I), a targeting molecule and a load.

[0335] In yet another aspect, a conjugate having the structure of formula (III) is provided:

[0336]

[0337] in

[0338] n, d, Ld1 and Ld2 are as defined in formula (I);

[0339] Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P;

[0340] M is hydrogen or LKa-LKb―P;

[0341] Provided that Q and M are not both hydrogen;

[0342] P is a carrier, and the carrier is connected to the B part or L of the compound of formula (I) 1 part;

[0343] A is an anti-Trop2 antibody or an antigen-binding fragment thereof, which is linked to G of the compound of formula (I) n part; G is glycine;

[0344] z is an integer of 1-20.

[0345] In one embodiment, LKa and LKb are as defined in formula (I).

[0346] In one embodiment, the conjugate has an integer or non-integer drug to antibody ratio (DAR) of 1-20.

[0347] As defined above, in one embodiment, G of the compound of formula (I) n The moiety is a recognition sequence for a ligase acceptor substrate, which facilitates the enzymatic coupling of the compound of formula (I) with the targeting molecule under the catalysis of the ligase. The targeting molecule is optionally modified and comprises a corresponding recognition sequence for a ligase acceptor or donor substrate.

[0348] It should be understood that when the antibody (or antigen-binding fragment) is combined with the G of the compound of formula (I) n When the moieties are conjugated under the catalysis of a ligase, the recognition sequence of the ligase acceptor substrate and the recognition sequence of the ligase donor substrate react with each other and form the resulting sequence.

[0349] In one embodiment, the antibody (or antigen-binding fragment) comprises LPXTGJ (SEQ ID NO: 49) as a recognition sequence for the ligase donor substrate, wherein J is as defined above. n During conjugation, the peptide bond upstream of the glycine in the LPXTGJ (SEQ ID NO: 49) sequence is cleaved by sortase A and the resulting intermediate is ligated to G n The free N-terminus of the peptide is cleaved to create a new peptide bond. The resulting sequence is LPXTG n(SEQ ID NO:57). Sequence G n and LPXTGJ (SEQ ID NO: 49) are as defined above.

[0350] In one embodiment, P is connected to the B part or L of the compound of formula (I) 1 part, and A is connected to G of the compound of formula (I) n Part of the compound is formed.

[0351] As defined above, R 10 will not appear in the B-P structure of the compound of formula (III). As defined above, when B in the compound of formula (I) is a terminal group R 10 When B is not present in the B-P structure of the compound of formula (III).

[0352] As defined above, in the compound of formula (III), A-G n In the structure, A optionally comprises a corresponding sequence generated by the reaction of the recognition sequence of the ligase acceptor substrate with the recognition sequence of the ligase donor substrate.

[0353] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein P is a load, the load is connected to the B part or L of the compound of formula (I) 1 part; each B is independently a terminal group R 10 , or a combination of the following 1), 2) and 3): 1) a self-removing spacer Sp1; 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; and 3) terminal group R 10 ; R 10 is hydrogen, or a group that can leave when reacting with a group in the supported substance; R 10 It represents the structural part that will not appear in the product molecule produced by the reaction of B and the load.

[0354] In one embodiment, M is hydrogen or LKa-L 2 ―L 1 ―B―P; wherein each B is independently absent, or is a combination of the following 1) and 2): 1) a self-resectable spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C4-10 Cycloalkylene, C 4-10 In a 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 In ―B―P, B does not exist. In one embodiment, in LKa-L 2 ―L 1 In ―B―P, B is a combination of 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 In one embodiment, in LKa-L 2 ―L 1 In ―B―P, B is -NH-CH2-U- or -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 connected to the load via an amide bond, an ester bond or an ether bond. In one embodiment, M is LKa-L 2 ―L 1 ―B―P, and B does not exist; and M can also be expressed as LKa-L 2 ―L 1 ―P.

[0355] Targeting molecules

[0356] In one embodiment, the targeting molecule is an anti-Trop2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ),in

[0357] The V H Include:

[0358] (i) a HCDR1 comprising 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;

[0359] (ii) a HCDR2 comprising 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;

[0360] (iii) comprising the amino acid sequence X 10 HCDR3 of GFGSSYWYFDV (SEQ ID NO: 3), wherein X 10 is G or S;

[0361] and / or

[0362] The V L Include:

[0363] (i) LCDR1 comprising the amino acid sequence KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0364] (ii) LCDR2 comprising the amino acid sequence SASYRYT (SEQ ID NO: 15); and

[0365] (iii) LCDR3 comprising the amino acid sequence QQHYITPLT (SEQ ID NO: 16).

[0366] In one embodiment, HCDR1 comprises the amino acid sequence NYGMN, TAGMQ, AAGMN or NAGMN.

[0367] In one embodiment, HCDR2 comprises amino acids WINTYTGEPTYTDDFKG (SEQ ID NO: 8), WINTHSGVPKYAEDFKG (SEQ ID NO: 9), WINTDSGEPTYTDDFKG (SEQ ID NO: 10).

[0368] In one embodiment, HCDR3 comprises amino acids GGFGSSYWYFDV (SEQ ID NO: 11) or SGFGSSYWYFDV (SEQ ID NO: 12).

[0369] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ) and the light chain variable region (V L ),in

[0370] The V H Include:

[0371] (i) a HCDR1 comprising the amino acid sequence X1AGMN (SEQ ID NO: 56), wherein X1 is N or A;

[0372] (ii) HCDR2 comprising the amino acid sequence WINTDSGEPTYTDDFKG (SEQ ID NO: 10);

[0373] (iii) a HCDR3 comprising the amino acid sequence GGFGSSYWYFDV (SEQ ID NO: 11);

[0374] and / or

[0375] The V L Include:

[0376] (i) LCDR1 comprising the amino acid sequence KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14);

[0377] (ii) LCDR2 comprising the amino acid sequence SASYRYT (SEQ ID NO: 15);

[0378] (iii) LCDR3 comprising the amino acid sequence QQHYITPLT (SEQ ID NO: 16).

[0379] In one embodiment, the V H Include:

[0380] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4,

[0381] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and

[0382] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11;

[0383] and / or

[0384] The V L Include:

[0385] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0386] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0387] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0388] In some embodiments, the V H Include:

[0389] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 5,

[0390] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and

[0391] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 12;

[0392] and / or

[0393] The V L Include:

[0394] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 14,

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

[0396] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0397] In some embodiments, the V H Include:

[0398] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4,

[0399] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and

[0400] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11;

[0401] and / or

[0402] The V L Include:

[0403] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0404] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0405] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0406] In some embodiments, the V H Include:

[0407] (i) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 7,

[0408] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, and

[0409] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11;

[0410] and / or

[0411] The V L Include:

[0412] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0413] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0414] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0415] In some embodiments, the V H Include:

[0416] (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 6,

[0417] (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and

[0418] (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11;

[0419] and / or

[0420] The V L Include:

[0421] (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13,

[0422] (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and

[0423] (iii) LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0424] In some embodiments, the V HIt comprises the following structure: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4, wherein FR1 comprises the amino acid of SEQ ID NO: 17, FR2 comprises the amino acid of SEQ ID NO: 18, FR3 comprises the amino acid of SEQ ID NO: 19, and FR4 comprises the amino acid of SEQ ID NO: 20.

[0425] In some embodiments, the V H comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NOs: 21-25. In some embodiments, the V H Contains the amino acid sequence of SEQ ID NO: 21. In some embodiments, the V H Contains the amino acid sequence of SEQ ID NO: 22. In some embodiments, the V H Contains the amino acid sequence of SEQ ID NO: 23. In some embodiments, the V H Contains the amino acid sequence of SEQ ID NO: 24. In some embodiments, the V H Comprising the amino acid sequence of SEQ ID NO:25.

[0426] In some embodiments, the V L comprising 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 some embodiments, the V L Contains the amino acid sequence of SEQ ID NO: 26. In some embodiments, the V L Comprising the amino acid sequence of SEQ ID NO:27.

[0427] In some embodiments, the V H comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24; and / or said V L Comprising 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.

[0428] In some embodiments, the V H Comprising the amino acid sequence of SEQ ID NO: 21, said V L Contains the amino acid sequence of SEQ ID NO: 26. In some embodiments, the V H Comprising the amino acid sequence of SEQ ID NO: 22, said V LContains the amino acid sequence of SEQ ID NO: 27. In some embodiments, the V H Comprising the amino acid sequence of SEQ ID NO: 23, said V L Contains the amino acid sequence of SEQ ID NO: 26. In some embodiments, the V H Comprising the amino acid sequence of SEQ ID NO: 24, said V L Contains the amino acid sequence of SEQ ID NO: 26. In some embodiments, the V H Comprising the amino acid sequence of SEQ ID NO: 25, said V L Comprising the amino acid sequence of SEQ ID NO:26.

[0429] In one embodiment, the antibody or antigen-binding fragment comprises: a heavy chain constant domain (CH), wherein the CH comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 28; and / or

[0430] 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.

[0431] In one embodiment, the antibody or antigen-binding fragment comprises: a heavy chain constant domain (CH), wherein the CH comprises the amino acid sequence of SEQ ID NO: 28, and

[0432] A light chain constant domain comprising the amino acid sequence of SEQ ID NO: 29.

[0433] In one embodiment, the antibody or antigen-binding fragment has an equilibrium dissociation constant (K) of about 0.5 nM to about 20 nM. D ) binds to TROP2. In one embodiment, K D The 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 ​​(including the endpoint values). In one embodiment, K D The values ​​are from about 7.5 nM to about 13.5 nM.

[0434] In one embodiment, the targeting molecule is an anti-human TROP2 antibody or an antigen-binding fragment thereof.

[0435] In one embodiment, the antibody is a recombinant antibody selected from 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 a scFv, a mini antibody, a bifunctional antibody, and a nano antibody.

[0436] 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 with the amino acid sequence of SEQ ID NOs: 30-33; and / or

[0437] 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.

[0438] At least about 90% is about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or a range between any two values, including the endpoints.

[0439] In one embodiment, the antibody or antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NOs: 30-33; and / or;

[0440] A light chain comprising the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:35.

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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.

[0445] Examples of anti-human TROP2 antibodies include, but are not limited to, Trodelvy's antibody (hRS7) and DS1062's antibody (Datopotamab). The antibody sequences are shown in Table 1.

[0446] In one embodiment, the pharmaceutical composition comprises an antibody or antigen-binding fragment.

[0447] Table 1 Antibody sequences

[0448]

[0449]

[0450]

[0451] Antibodies conjugated to linker-cargo

[0452] In order to be conjugated with the compound of formula (I), the antibody herein may include a modified portion to be connected to the Gn in the compound of formula (I). The position of introduction of such a modified portion is not limited, for example, its introduction position may be, but is not limited to, at the C-terminus or N-terminus of the antibody heavy chain or light chain.

[0453] In one embodiment, the conjugate herein formed by conjugating an anti-human TROP2 antibody to a load can specifically bind to TROP2 on the surface of tumor cells and selectively kill tumor cells expressing TROP2. In another preferred embodiment, the use of the conjugate herein or the pharmaceutical composition herein in the manufacture of a medicament for treating a disease, disorder or condition selected from a TROP2-positive tumor is provided. In a more preferred embodiment, the disease, disorder or condition is selected from breast cancer, urothelial carcinoma, lung cancer, liver cancer, endometrial cancer, head and neck cancer, ovarian cancer, etc.

[0454] In an alternative embodiment, the modified moiety conjugated to Gn in the compound of formula (I) can be introduced into a non-terminal position of the antibody heavy or light chain using, for example, chemical modification methods.

[0455] In one embodiment, the targeting molecule herein is an antibody or an antigen binding fragment thereof, which may include terminal modifications. Terminal modifications refer to modifications at the C-terminus or N-terminus of an antibody heavy chain or light chain, and the modifications, for example, include a ligase recognition sequence. In another embodiment, the terminal modifications may also include a spacer Sp2 containing 2-100 amino acids, wherein the antibody, Sp2, and ligase recognition sequences are sequentially connected. In a preferred embodiment, Sp2 is a spacer sequence containing 2-20 amino acids. In a specific embodiment, Sp2 is a spacer sequence selected from the following: GA, GGGGS (SEQ ID NO: 42), GGGGSGGGGS (SEQ ID NO: 43), and GGGGSGGGGSGGGGS (SEQ ID NO: 44), especially GA.

[0456] In a preferred embodiment, the light chain of the antibody or antigen-binding fragment thereof includes three types: wild type (LC); C-terminally modified light chain (LCCT), which is modified by directly introducing the ligase recognition sequence LPXTG (SEQ ID NO: 48); and C-terminally modified light chain (LCCT L ), which is modified by introducing a short peptide spacer plus a ligase donor substrate recognition sequence LPXTG (SEQ ID NO: 48). The heavy chain of the antibody or its antigen-binding fragment includes three types: wild type (HC); C-terminally modified heavy chain (HCCT), which is modified by directly introducing a ligase recognition sequence LPXTG (SEQ ID NO: 48); and C-terminally modified heavy chain (HCCT L ), which is modified by introducing a short peptide spacer plus a ligase donor substrate recognition sequence LPXTG (SEQ ID NO: 48). X can be any natural or unnatural single amino acid. In one embodiment, X is glycine. The sequence of the modified antibody is shown in Table 2.

[0457] The conjugates herein may further comprise a load. The load is as described above.

[0458] In one embodiment, the pharmaceutical composition comprises an antibody-drug conjugate (ADC).

[0459] Table 2 Sequences of modified antibodies

[0460]

[0461]

[0462] Specific examples of conjugates

[0463] In one embodiment, Q is hydrogen, each LKa is In one embodiment, Formula (III) has the structure of Formula (III-a):

[0464]

[0465] wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0466] In one embodiment, Ld2 is a chemical bond and d is 0. In one embodiment, the compound of formula (III-a) is as follows:

[0467]

[0468] In one embodiment, d is 0, Ld2 is In one embodiment, the compound of formula (III-a) is as follows:

[0469]

[0470] 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:

[0471]

[0472]

[0473] In one embodiment, Ld2 is d is 0. In one embodiment, the compound of formula (III-a) is as follows:

[0474]

[0475] In one embodiment, d is 1, 2 or 3, and Ld2 is and each Ld1 is independently selected from In one embodiment, the compound of formula (III-a) is as follows:

[0476]

[0477] 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.

[0478] In one embodiment, LKa is In one embodiment, Formula (III) has the structure of Formula (III-b):

[0479]

[0480] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0481] In one embodiment, Q is hydrogen, d is 1, and Ld2 is And Ld1 is selected from In one embodiment, the compound of formula (III-b) is as follows:

[0482]

[0483] In one embodiment, Q is -C2H4-(PEG) t -(CO)NH2, d is 1, Ld2 is a chemical bond, and Ld1 is selected from In one embodiment, the compound of formula (III-b) is as follows:

[0484]

[0485] 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.

[0486] In one embodiment, B in the compound of formula (I) is a terminal group R 10 , and L 1The cleavable sequence 1 in the formula is connected to the load to form a compound of formula (II), wherein B is not present in the molecule obtained by connecting the cleavable sequence 1 to the load. In this case, M can be understood as LKa-L2―L1―B―P, wherein B is not present. In this case, M can also be expressed as LKa-L 2 ―L 1 ―P. In one embodiment, n is 3, L 2 Yes - (CH2) p -(CH2)2(CO)-, p is 3, L 1 is GGFG, B is -NH-CH2-U- or absent, or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, U is O, g is 1.

[0487] In one embodiment, conjugate III-a-0-1 has the following structure:

[0488]

[0489] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0490] In one embodiment, conjugate III-a-0-2 has the following structure:

[0491]

[0492] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0493] In one embodiment, conjugate III-a-0-3 has the following structure:

[0494]

[0495] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0496] In one embodiment, conjugate III-a-0-4 has the following structure:

[0497]

[0498] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) nconnect.

[0499] In one embodiment, conjugate III-a-0-5 has the following structure:

[0500]

[0501] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0502] In one embodiment, conjugate III-a-1-1 has the following structure:

[0503]

[0504] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0505] In one embodiment, conjugate III-a-1-2 has the following structure:

[0506]

[0507]

[0508] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0509] In one embodiment, conjugate III-a-1-3 has the following structure:

[0510]

[0511] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0512] In one embodiment, conjugate III-a-1-4 has the following structure:

[0513]

[0514] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0515] In one embodiment, i is 4, g is 1, R 11 It's methyl.

[0516] In one embodiment, n is 3, L 2 Yes -(C2H4-O) p -(CH2)2(CO)-, p is 2, L 1 is GGFG, B is -NH-CH2-U-, and U is O. In one embodiment, conjugate III-b-1-1 has the following structure:

[0517]

[0518] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0519] In one embodiment, conjugate III-a-0-2 is as follows (conjugate III-a-0-2-1):

[0520]

[0521] wherein A is an anti-TROP2 antibody or antigen-binding fragment, and the antibody or antigen-binding fragment is modified to be linked to (Gly)n.

[0522] In one embodiment, i is 4, j is 8, g is 1, R 11 In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-1):

[0523]

[0524] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0525] In one embodiment, i is 4, j is 12, g is 1, R 11 In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-1):

[0526]

[0527] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0528] In one embodiment, i is 4, j is 12, R 11 In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-2):

[0529]

[0530] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0531] In one embodiment, i is 4, j is 12, R 11 In one embodiment, conjugate III-a-1-2 is as follows (conjugate III-a-1-2-4-3):

[0532]

[0533] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0534] In one embodiment, n is 3, i is 4, j is 12, m is 1, R 11 In one embodiment, conjugate III-b-1-1 is as follows (conjugate III-b-1-1-4):

[0535]

[0536] wherein A is an anti-TROP2 antibody or antigen-binding fragment, the antibody or antigen-binding fragment is modified to bind to (Gly) n connect.

[0537] Preparation of conjugates

[0538] The conjugates herein can be prepared by any method known in the art. In some embodiments, the conjugates are prepared by site-specific conjugation of a targeting molecule catalyzed by a ligase with a compound of formula (I) with a load, wherein the targeting molecule is modified by a ligase recognition sequence. The method comprises steps A and B.

[0539] Step A. Preparation of Linker-Loader Intermediate

[0540] In a preferred embodiment, B in the compound of formula (I) is covalently linked to a carrier having another reactive group through a reactive group.

[0541] The linker-carrier intermediate prepared using the compound of formula (I) herein has a defined structure, a defined composition and high purity, so that when the conjugation reaction is carried out with the antibody, less impurities or no other impurities are introduced. When such an intermediate is used for site-specific conjugation catalyzed by a modified antibody containing a ligase recognition sequence, a homogeneous ADC with highly controllable quality can be obtained.

[0542] Step B. Connecting the targeting molecule to the compound of formula (I) with a load

[0543] The targeting molecule herein can be conjugated to the compound of formula (I) (ie, the compound of formula (II)) with a load by any method known in the art.

[0544] The targeting molecule and the compound of formula (I) with a load are connected to each other through a substrate recognition sequence specific for a ligase. The recognition sequence depends on the specific ligase used. In one embodiment, the targeting molecule is an antibody that introduces a terminal modification based on a recognition sequence at the C-terminus of a light chain and / or a heavy chain; under the catalysis of a wild-type or optimized engineered ligase or any combination thereof and under suitable catalytic reaction conditions, the targeting molecule is conjugated to a compound of formula (II).

[0545] In a specific embodiment, the ligase is sortase A and the conjugation reaction can be represented by the following scheme:

[0546]

[0547] The triangle represents a portion of the antibody; and the pentagon represents a portion of the compound of formula (II). n, X and J are as defined above. n During conjugation, the peptide bond upstream of the glycine in the LPXTGJ (SEQ ID NO: 49) sequence is cleaved by sortase A and the resulting intermediate is ligated to G n The free N-terminus of the peptide is cleaved to create a new peptide bond. The resulting amino acid sequence is LPXTG n (SEQ ID NO:57). Sequence G n and LPXTGJ (SEQ ID NO: 49) are as defined above.

[0548] Metabolism of the conjugate in a physiological environment

[0549] When a part or the whole of the linker is cleaved in tumor cells, the anti-tumor compound is partially released to show the anti-tumor effect of the anti-tumor compound. Since the linker is cleaved at the connection position with the drug, the anti-tumor compound is released with its inherent structure to show its inherent anti-tumor effect.

[0550] In one embodiment, the cleavable sequence 1 (eg, GGFG) is cleavable by a lysosomal enzyme (eg, cathepsin B and / or cathepsin L).

[0551] In one embodiment, Sp1 comprises a self-immolative spacer. In one embodiment, Sp1 comprises PABC, acetal or heteroacetal. In one embodiment, L 1is 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 site and a self-cleaving spacer, which is cleaved in the cell and releases the target molecule (such as a drug).

[0552] Pharmaceutical compositions and pharmaceutical preparations

[0553] Another object of the present invention is to provide a pharmaceutical composition comprising a preventively or therapeutically effective amount of the conjugate herein and at least one pharmaceutically acceptable carrier.

[0554] Another object of the present invention is to provide a pharmaceutical composition comprising a preventively or therapeutically effective amount of the anti-TROP2 antibody herein, and at least one pharmaceutically acceptable carrier.

[0555] The pharmaceutical composition herein can be administered in any manner, as long as it achieves prevention, alleviation, prevention or cure of symptoms of humans or animals. For example, various suitable dosage forms can be prepared according to the route of administration (especially injection), such as lyophilized powder for injection, injection solution or sterile powder for injection.

[0556] The term "pharmaceutically acceptable" means that when in contact with the tissues of a patient within the scope of normal medical judgment, it should not cause excessive toxicity, irritation or allergic response, etc., has a reasonable advantage-disadvantage ratio and is effective for the intended purpose.

[0557] The term "pharmaceutically acceptable carrier" refers to a carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline, etc. Pharmaceutically acceptable carriers also include carrier materials that allow the composition to approach physiological conditions, such as pH regulators, buffers, toxicity regulators, etc., as well as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In some embodiments, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with a therapeutic active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. 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, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition may also contain a small amount of a wetting agent, emulsifier, or pH buffer such as acetate, citrate or phosphate.

[0558] In one embodiment, the pharmaceutical composition of the present disclosure has an integer or non-integer drug to antibody ratio (DAR) of 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 specific embodiment, the DAR of the conjugate of the present disclosure is about 2, about 4, about 6, or about 8.

[0559] Treatments and uses

[0560] The conjugate or antibody can be used to treat tumors and / or autoimmune diseases. Tumors sensitive to conjugate treatment include tumors characterized by specific tumor-associated antigens or cell surface receptors, and the specific tumor-associated antigens or cell surface receptors will be recognized by the targeting molecule in the conjugate and can be killed by the cargo / cytotoxin in the conjugate.

[0561] Therefore, in yet another aspect, there is also provided use of the conjugate or antibody herein or the pharmaceutical composition herein in the manufacture of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0562] In another aspect, a conjugate herein or a pharmaceutical composition herein for use in treating a tumor or an autoimmune disease is provided.

[0563] In another aspect, an antibody herein or a pharmaceutical composition herein for use in treating a tumor or an autoimmune disease is provided.

[0564] In another aspect, a method for treating a tumor or an autoimmune disease is provided, the method comprising administering an effective amount of the conjugate or antibody herein or the pharmaceutical composition herein to an individual in need thereof.

[0565] In a preferred embodiment, the conjugates herein formed by conjugating anti-human TROP2 antibodies to small molecule cytotoxins can specifically bind to TROP2 on the surface of tumor cells and selectively kill tumor cells expressing TROP2. In another preferred embodiment, there is provided the use of the conjugates (or antibodies) herein or the pharmaceutical compositions herein in the manufacture of a medicament for treating a disease, disorder or condition selected from a TROP2-positive tumor. In a more preferred embodiment, the disease, disorder or condition is a TROP2-positive tumor. In one embodiment, TROP2-positive tumors are selected from the group consisting of: breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, etc.

[0566] The dosage of the conjugate (or antibody) administered to a subject can be adjusted over a wide range. The dosage can vary according to the specific route of administration and the needs of the subject, and can be determined by a health care professional.

[0567] Beneficial Effects

[0568] The antibody-drug conjugate of the present invention uses a specially designed linker-load and is more stable and can achieve great efficacy at a lower DAR, and thus can reduce side effects and increase the therapeutic index.

[0569] This paper utilizes a linking unit with a unique structure and uses a ligase to catalyze the conjugation of a targeting molecule with a load. The conjugate herein has good homogeneity, high activity and high selectivity. In addition, the toxicity of the linking unit-load intermediate is much lower than that of the free load, and therefore, the manufacturing process of the drug has no obvious disadvantages, which will be beneficial to industrial production.

[0570] The conjugates herein achieve at least one of the following technical effects:

[0571] (1) High inhibitory activity against target cells or strong killing effect on target cells.

[0572] (2) Good physicochemical properties (e.g. solubility, physical and / or chemical stability).

[0573] (3) Good pharmacokinetic properties (e.g., good stability in plasma, appropriate half-life and duration of action).

[0574] (4) Good safety (low toxicity to non-target normal cells or tissues, and / or fewer side effects, wider therapeutic range), etc.

[0575] (5) Highly modular design and simple assembly of multiple drugs.

[0576] Example

[0577] Preparation Example

[0578] In order to more clearly illustrate the purpose and technical solutions, the present invention will be further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of this invention. Specific experimental methods not mentioned in the following examples are carried out according to conventional experimental methods.

[0579] Instruments, materials, and reagents

[0580] Unless otherwise stated, the instruments and reagents used in the examples are commercially available. The reagents were used directly without further purification.

[0581] MS: Thermo Fisher Q Exactive Plus, Water2795-Quattro micro triple quadrupole mass spectrometer HPLC: Waters 2695, Agilent 1100, Agilent 1200

[0582] Semi-preparative HPLC: Lisure HP plus 50D

[0583] Flow cytometer: CytoFLEX S

[0584] HIC-HPLC: Butyl-HIC; mobile phase A: 25mM PB, 2M (NH4)2SO4, pH 7.0; mobile phase B: 25mM PB, pH 7.0; flow rate: 0.8ml / min; acquisition time: 25 minutes; injection volume: 20μg; column temperature: 25℃; detection wavelength: 280nm; sample chamber temperature: 8℃.

[0585] SEC-HPLC: column: TSK-gel G3000 SWXL, TOSOH 7.8mm ID×300mm, 5μm; mobile phase: 0.2M KH2PO4, 0.25M KCl, pH 6.2; flow rate: 0.5ml / min; acquisition time: 30 minutes; injection volume: 50μl; column temperature: 25℃; detection wavelength: 280nm; sample plate temperature: 8℃.

[0586] CHO was obtained from Thermo Fisher Scientific; pcDNA 3.3 was obtained from Life Technology; HEK293F was obtained from Prejin; PEIMAX transfection reagent was obtained from Polyscience; MabSelect Sure ProA was obtained from GE; Capto S ImpAct was obtained from GE; Rink-amide-MBHA-resin and dichlororesin were obtained from Nankaisynthesis; HCC1954 was obtained from ATCC catalog number CRL-2338; SK-BR-3 was obtained from ATCC (catalog number HTB-30); BT-474 was obtained from ATCC (catalog number HTB-20); NCI-N87 cells were obtained from ATCC (catalog number CRL-5822); MCF7 was obtained from ATCC (catalog number HTB-22); MDA-MB-231 was obtained from ATCC (catalog number HTB-26); MDA-MB-468 was obtained from ATCC (catalog number HTB-132); CFPAC-1 was obtained from ATCC (catalog number CRL-1918); NCI-H2110 was obtained from ATCC (catalog number CRL-5924); JIMT-1 was obtained from Wuxi Apptech; Capan-1 was obtained from ATCC (Catalog No. CRL-1573); the optimized recombinant enzyme sortase A from Staphylococcus aureus was produced in E. coli.

[0587] Example 1 Construction of antibody expression vector, antibody expression, purification and identification

[0588] 1.1 Construction of expression vector encoding anti-TROP2 antibody

[0589] To produce an expression vector encoding the anti-TROP2 antibody light chain, the nucleic acid sequence of LC was individually cloned into the pCDNA3.3 vector (Life technology); to produce an expression vector encoding the anti-TROP2 antibody heavy chain, the nucleic acid sequence of HC was individually cloned into the pCDNA 3.3 vector (Life technology).

[0590] Table 3 Antibody sequences

[0591]

[0592] Note: During the preparation of ADC, the upstream peptide bond of GG in the LPETGG (SEQ ID NO: 51) sequence was cleaved by sortase A, and the resulting intermediate was ligated to the free N-terminus of G3 in the linker-payload to generate a new peptide bond.

[0593] 1.2 Expression of anti-TROP2 antibodies

[0594] The plasmids encoding the light and heavy chains of the anti-TROP2 antibody were paired and mixed at a mass ratio of 2: 1. The plasmid pairs and PEIMAX (Polyscience) transfection reagent were diluted in HEK293F basal medium, respectively, and then mixed evenly. The mixture was allowed to stand at room temperature and added to the HEK293F seed cell culture. The cells were cultured at 32 degrees for 24 hours, sampled for cell density and viability analysis, and supplemented with 10% by volume of HEK293F feed medium. Then, the culture temperature was converted to 32°C for subsequent culture. At 72 hours of cultivation, the cell culture was sampled again for cell density and viability analysis. At 144 hours of cultivation, the cell culture was sampled for cell density and viability analysis.

[0595] 1.3 Purification of anti-TROP2 antibodies

[0596] Following the manufacturer's instructions, antibodies are purified by affinity chromatography. Briefly, MabSelect SureLX resin (GE Healthcare) is loaded into a chromatographic column (BestChrom, Shanghai, China) and balanced with 50mM Tris, 150mM NaCl (pH 7.4). Then, cell culture supernatant is obtained and applied to the column. The column is washed with 50mM Tris, 150mM NaCl (pH 7.4) to remove non-specifically bound proteins. Then, the antibody is eluted with a 50mM citrate buffer at pH 3.5, and 1M Tris-HCl (pH 9.0) is used to adjust the eluent containing the antibody to pH 6.5. Finally, the buffer of the antibody is exchanged into 50mM Tris, 150mM NaCl, pH 7.4 by an Anicon Ultra-15 centrifugal filter (Merk Millipore).

[0597] 1.4 Binding kinetics and affinity analysis

[0598] Binding kinetics and affinity analyses were performed. Surface plasmon resonance (SPR) analyses were performed on a Biacore T200 (GE healthcare) with sensor chip Protein A (GE healthcare) following the manufacturer's instructions. All measurements were performed at 25°C in HBS-EP +The assay was performed in a buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). About 110-140 RU (resonance units) of each purified antibody were captured on flow cells 2 and 4 (i.e., reaction surfaces) of the sensor chip, respectively. Flow cells 1 and 3 were washed with HBS-EPES. + Buffer treatment to serve as a reference surface.

[0599] In HBS-EP + Serial dilutions of the recombinant extracellular domain of human TROP2 (i.e., analyte) (Acrobio system) (243, 81, 27, 9, 3, 1, 0.333 and 0.111 nM, respectively) were prepared in buffer. The capture antibody was followed by a three-minute injection of the TROP2 serial dilutions at a flow rate of 30 μL / min (association phase) and a ten-minute buffer flow (dissociation phase). The chip surface was regenerated by two 30-second pulse injections of 10 mM glycine hydrochloride (pH 1.5) at a flow rate of 50 μL / min. The collected data were processed on the Biacore T200 evaluation software using methods known in the art, including the following steps: (1) setting the reaction on the y-axis and the injection starting point on the x-axis to zero; (2) performing double referencing by first subtracting the reference surface data from the reaction surface data to obtain the analyte injection curve, and then subtracting the buffer injection curve from the analyte injection curve; and (3) performing kinetic analysis using a 1:1 binding model with global fitting. The results for each antibody are presented as Ka (association rate), Kd (dissociation rate) and K D (equilibrium dissociation constant) is presented.

[0600] 1.5 SEC-HPLC detection of anti-TROP2 antibodies

[0601] The antibody samples were centrifuged at 12,000 rpm for 5 minutes, and the supernatant was applied to a SEC-HPLC column to detect the percentage of monomeric form (corresponding to intact antibody), high molecular weight form (HMW, corresponding to antibody aggregates resulting from aggregation), and low molecular weight form (LMW, corresponding to antibody fragments resulting from degradation) of each antibody.

[0602]

[0603] Trop2 is widely expressed in normal tissues, and the affinity of Ab13 and Ab16 is reduced. Low-affinity antibodies should be able to improve safety while maintaining efficacy, which has been verified in existing experiments.

[0604] 1.6 Internalization activity

[0605] MDA-MB-468 cells with good viability were treated with trypsin, collected, suspended in cold FACS buffer (DPBS + 2% FBS), and adjusted to 2×10 6 cells / ml. Anti-Trop2 antibody and isotype control antibody samples were fluorescently labeled by mixing with anti-human IgG-Fc-AF647 secondary antibody at a 1:1 molar ratio for 20 minutes at room temperature. The labeled antibody was added to the cell suspension at a final concentration of 10μg / 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. The cells were incubated at 37°C for 0 minutes, 10 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, and 240 minutes for antibody internalization, and at each time point, 1×10 5 Cells / well were transferred to a V-bottom 96-well plate. Internalization was stopped by placing the cells back in an ice bath. Non-internalized cells (0 minutes) were divided into MAX and MIN groups, while cells internalized by the antibody were labeled as I groups. 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.

[0606] The MFI data were substituted into the following formula and the results were analyzed using a one-phase exponential correlation function in Prism 6.

[0607] Isotype control: MFI (I) =MFI (样品，I) -MFI (同型对照抗体，I)

[0608] MFI (MAX) =MFI (样品，MAX) -MFI (阴性抗体，MAX)

[0609] MFI (MIN) =MFI (样品，MIN) -MFI (阴性抗体，MIN)

[0610] Internalization ratio: R (t) =(MFI (I) -MFI (MIN) ) / MFI (MAX) ×100%.

[0611] like Figure 1.1 As shown in , all four antibodies showed similar internalization activity on MDA-MB-468.

[0612] Example 2 Preparation of intermediates

[0613] Example 2.1 Preparation of Linker-Loader 1 and Linker-Loader 2

[0614]

[0615] opSu is mixture of

[0616] Preparation of intermediate MC-GGFG-DXd

[0617] The intermediate MC-GGFG-DXd is either commercially available or prepared following the procedure described in EP 2907824. This compound was used to prepare Linker-Loader 1.

[0618] Preparation of linker-load intermediate 1

[0619]

[0620] The linker-load intermediate 1 can be synthesized by conventional solid phase peptide synthesis using Rink-amide-MBHA-resin. The amino acids in the linker unit are protected using Fmoc. The coupling reagent is selected from HOBT, HOAt / DIC, DCC, EDCI or HATU. After synthesis, the product is cleaved from the resin using TFA / TIS / H2O solution. The product is purified by HPLC, lyophilized and stored for future use. MS m / z: [MH] - =1382.6.

[0621] Preparation of linker-carrier 1

[0622] The linker-load intermediate 1 and MC-GGFG-DXd (molar ratio of about 1:2) were weighed and dissolved in water and DMF, respectively, and then mixed thoroughly to obtain a mixture, and the mixture was reacted at 0-40°C for 0.5-30 hours. After the reaction was completed, an appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and the reaction was continued at 0-40°C for another 0.2-20 hours. After the reaction was completed, the product was purified by semi-preparative / preparative HPLC and lyophilized to obtain the linker-load 1. MS m / z: [(M+3H) / 3] + =1163.3.

[0623] Preparation of linker-carrier 2

[0624] The following linker-payload 2 can be prepared using similar synthetic routes and reagents as linker-payload 1.

[0625]

[0626] opSu is mixture.

[0627] Example 2.2 Preparation of Linker-Loader 3 and Linker-Loader 4

[0628] Preparation of intermediate 11

[0629]

[0630] 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 addition of 2-bromo-5-fluoroaniline (100 g, 526.27 mmol) in portions at room temperature. The mixture was stirred for 3 hours and then poured into 2000 mL of ice-water. A precipitate was 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 HNMR (400MHz, 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).

[0631] Step B: N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide: To a stirred solution of N-(2-bromo-5-fluorophenyl)acetamide (105 g, 452.48 mmol) in THF (1000 mL) was added dropwise n-BuLi (594 mL, 1.6 M in n-hexane, 950.22 mmol) at -78 °C over 1 hour. After completion, the mixture was stirred for 0.5 hours under N2. Then, at -78 °C, a solution of cyclobutanone (38.06 g, 542.98 mmol) in THF (50 mL) was added dropwise over 0.5 hours, and the mixture was stirred at -78 °C to room temperature for 6 hours. At 0 °C, the mixture was poured into 500 mL of saturated NH4Cl aqueous solution. It was extracted with ethyl acetate (500 mL×3), washed with brine (250 mL×2), dried over Na2SO4 and concentrated. The mixture was triturated with (PE / EA=1:1, 100 mL) for 10 minutes, filtered and the filter cake was collected and dried in vacuo to give N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g) as a yellow solid. LCMS m / z 206.1 (M-18+H), 246.1 (M+Na).

[0632] Step C: N-(3-Fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirred mixture of N-(5-fluoro-2-(1-hydroxycyclobutyl)phenyl)acetamide (24 g, 107.50 mmol) in CH2Cl2 (170 mL) and water (170 mL) were added silver nitrate (AgNO3) (5.48 g, 32.25 mmol) and potassium persulfate (K2S2O8) (58.12 g, 215.01 mmol) and the mixture was stirred at 30°C for 6 hours. The mixture was filtered on 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-1-yl)acetamide (14 g) as a light yellow solid. MS m / z 222.1 (M+H).

[0633] Step D: N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide: To a stirring mixture of N-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14 g, 63.28 mmol) in THF (500 mL) at 0°C was added 1-butyl nitrite (8.48 g, 63.28 mmol), followed by t-BuOK (8.52 g, 75.94 mmol). The mixture was stirred at 0°C for 2 hours. Upon completion, the mixture was acidified with HCl (2N) to adjust to pH = 3. The mixture was extracted with ethyl acetate (200 mL×3), washed with brine (100 mL×2), dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was triturated with tert-butyl methyl ether (200 mL) for 10 minutes, filtered and the filter cake collected and dried in vacuo to give N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (12 g) as a yellow solid. MS m / z 251.1 (M+H).

[0634] Step E: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide: To a solution of N-(3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (12 g, 47.96 mmol) in acetic anhydride (90 mL) and THF (90 mL) was added 10% Pd / C (1 g), and the mixture was stirred at 25°C for 16 hours under H2 atmosphere. After cooling to 0°C, Et3N (20 mL) was added dropwise, and the mixture was stirred at 0°C for 1 hour. Filtered on celite, the filtrate was poured into ice-water (500 mL). Extracted with ethyl acetate (500 mL×3), washed with brine (250 mL×2), dried over Na2SO4 and concentrated. The residue was triturated with tert-butyl methyl ether (120 mL) for 10 minutes, filtered and the filter cake collected and dried in vacuo to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7.9 g) as a yellow solid. MS m / z 279.1 (M+H).

[0635] Step F: N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide: To a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7.9 g, 28.39 mmol) in MeOH (150 mL) was added aqueous HCl (2N, 150 mL), and the mixture was stirred at 50 ° C for 7 hours. After cooling to 0 ° C, saturated aqueous NaHCO3 was added dropwise to adjust to pH = 8. Extracted with ethyl acetate (200 mL×3), washed with brine (200 mL×2), dried over Na2SO4 and concentrated under reduced pressure to give N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (6.0 g) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 6.57 (s, 3H), 6.18 (td, J = 11.1, 2.4 Hz, 2H), 4.52 (dt, J = 13.3, 5.0 Hz, 1H), 3.13 (ddd, J = 17.5, 13.0, 4.6 Hz, 1H), 3.00-2.81 (m, 1H), 2.69 (dtd, J = 9.4, 4.6, 2.5 Hz, 1H), 2.09 (s, 3H), 1.79 (qd, J = 13.0, 4.3 Hz, 1H). MS m / z 237.1 (M+H).

[0636] Step G: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide: NCS (2.26 g, 16.93 mmol) was added in batches to a solution of N,N'-(3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl) diethylamide (4.0 g, 16.93 mmol) in DMF (80 mL) 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 was formed, which was collected by filtration and dried in vacuo at room temperature to give N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide (4.0 g) as a yellow solid. 1 HNMR (400MHz, DMSO-d6) δ8.11(d,J=8.0Hz,1H),7.71(s,2H),6.62(d,J=11.9Hz,1H),4.53(ddd,J=13.0,8.0 ,4.7Hz,1H),3.18-3.04(m,1H),2.91(ddd,J=17.5,12.4,4.8Hz,1H),2.21-2.08(m,1H),1.99-1.83(m,4H). MS m / z271.0(M+H).

[0637] Step H: N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide: N-(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (4.0 g) was added. To a mixture of (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (4.28 g, 16.25 mmol), pyridinium p-toluenesulfonate (1.11 g, 4.43 mmol) and o-cresol (10 mL) were added, and the mixture was heated to reflux under N2 for 24 h. The solvent was removed under reduced pressure and the mixture was purified by FCC (THF / CH2Cl2=0-60%) to give N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (4.1 g) as a brown solid. MS m / z 498.1 (M+H).

[0638] 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 concentrated aqueous HCl was stirred at 70 °C under N2 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).

[0639] Preparation of intermediate 12 (12-1, 12-2)

[0640]

[0641] 12-1 and 12-2 were 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.

[0642]

[0643] The above HPLC conditions: equipment: Agilent 1200; column: Waters XBridge C18 4.6*50mm, 3.5um; flow rate: 2.0mL / min; elution gradient: 5.0%-95.0%-95.0%-5.0%-5.0%, 0.00min-1.50min-2.50min-2.52min-3.00min; temperature: 40°C; phase: A: acetonitrile; B: H2O (0.05% TFA); wavelength: 214nm / 254nm.

[0644] Preparation of linker-carrier 3

[0645]

[0646] opSu is mixture.

[0647] Preparation of Compound 13 (Step A)

[0648] Weigh 4.33g Fmoc-Gly-Gly-OH and 6.84g Pb(OAc)4 and add to a 500ml single-necked round-bottom flask. Under a nitrogen atmosphere, add anhydrous THF / toluene (120 / 40ml) and stir to dissolve. Then, 1.16mL pyridine is added to the reaction system. The reaction system is heated to 80°C and refluxed for 5 hours under a nitrogen atmosphere. Samples are obtained and detected by HPLC to monitor the reaction.

[0649] The reaction system was cooled to room temperature, filtered, and the filter cake was washed with EA three times. The filtrates were combined and concentrated to dryness. Column chromatography (PE:EA=100:0-50:100) was performed to obtain about 2000 mg of the target product as a white solid with a yield of 44%.

[0650] Preparation of Compound 15 (Step B)

[0651] 200 mg of compound 13 was weighed and added to a 100 ml single-neck round-bottom flask. Then, 15 ml of THF was added and stirred to dissolve. Then, compound 14 (312 mg, 3.0 eq) and TsOH·H2O (15 mg, 0.15 eq) were added to the reaction system. The reaction system was allowed to react overnight at room temperature. A sample was obtained and detected by TLC (PE / EA=1:1) to monitor the reaction. The raw material was substantially disappeared, and a new spot was detected.

[0652] Saturated sodium bicarbonate solution was added to quench the reaction. Extracted 3 times with EA. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by column chromatography (PE:EA=5:1-1:1) to give about 80 mg of the target product as a colorless oil with a yield of 29%. MS m / z: [M+H] + =501.1

[0653] Preparation of Compound 16 (Step C)

[0654] 200mg of compound 15 was weighed and added to a 100ml single-necked round-bottom flask. Then, 10ml of EtOH and 5ml of EA were added to completely dissolve. Then, under a nitrogen atmosphere, 40mg of palladium carbon was added to the reaction system, and the reaction system was purged with hydrogen three times. The reaction system was kept under a hydrogen atmosphere and stirred at room temperature for 0.5 hours. A sample was obtained and detected by TLC (DCM / MeOH=10:1) to monitor the reaction. The raw material essentially disappeared, and a new spot was detected.

[0655] The reaction system was filtered and the filter cake was washed with EA 3 times. The filtrates were combined and concentrated to dryness to obtain 200 mg of the product as a white solid with a yield of 100%. The product was directly used in the next reaction without purification. MS m / z: [MH] - =409.4.

[0656] Preparation of Compound 21 (Step D)

[0657] Step D-1

[0658] Weigh 2.0g of dichlororesin and put it into a peptide synthesis tube. Add DCM (10ml) and allow it to swell at room temperature for 30 minutes. Remove the solvent by vacuum suction. Wash the resin twice with DCM, where the volume of each wash is 7mL and the time length is 1 minute. Remove the solvent by vacuum suction. Next, weigh compound 16 (200mg) and add it to a 50ml centrifuge tube. Add DCM (about 10ml). Dissolve the solid by shaking. Add to the above resin. Stir to soak 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 complete, add an appropriate amount of methanol. Stir for 30 minutes. Remove the solvent by vacuum suction. Wash the resin once with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, where the volume of each wash is 10mL and the time length is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin for ninhydrin detection. The resin was colorless and transparent, and the solution was slightly yellow, indicating suitability for the next coupling step.

[0659] Step D-2

[0660] Deprotection was performed twice by adding 10 mL of an existing 20% ​​piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, wherein the volume of each wash was 10 mL and the length of time was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0661] Add 563 mg of Fmoc-Phe-OH and 197 mg of HOBt to 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 obtain an activated reaction solution.

[0662] 3 molar equivalents of activated reaction solution are added to the resin. Stir so that the resin is fully immersed in the solution (if there is resin attached to the tube wall, a small amount of DCM is used to wash the tube wall). Stir 2-3 hours. After the reaction is complete, remove the solvent by vacuum suction. The resin is washed twice with DMF in sequence, methanol wash once, DMF wash once, methanol wash once and DMF wash twice, wherein the volume of each washing is 10mL and the time length is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin and carry out ninhydrin detection. The resin is colorless and transparent, and the solution is slightly yellow, showing that it is suitable for the next coupling step.

[0663] Step D-3

[0664] Deprotection was performed twice by adding 10 mL of an existing 20% ​​piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was complete, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, wherein the volume of each wash was 10 mL and the length of time was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue.

[0665] Add 531 mg of Fmoc-GG-OH and 197 mg of HOBt to a 50 mL centrifuge tube. Then, add about 10 mL of DMF. Dissolve the solid by shaking. Then, add 0.24 mL of DIC. Activate for 10-30 minutes to obtain an activated reaction solution.

[0666] 3 molar equivalents of activated reaction solution are added to resin. Stir so that resin is fully immersed in the solution (if there is resin attached to the tube wall, a small amount of DCM is used to wash the tube wall). Stir 2-3 hours. After the reaction is complete, remove the reaction solution by vacuum suction. Resin is washed twice with DMF in sequence, methanol wash once, DMF wash once, methanol wash once and DMF wash twice, wherein the volume of each washing is 10mL and the time length is 1 minute. Remove solvent by vacuum suction. Take a small amount of dry resin and carry out ninhydrin detection. Resin is colorless and transparent, and the solution is slightly yellow, shows that it is suitable for the next coupling step.

[0667] Step D-4

[0668] Deprotection was performed twice by adding 10 mL of existing 20% ​​piperidine / DMF solution and reacting for 10 minutes each time. After the reaction was completed, the solution was removed by vacuum suction. The resin was washed twice with DMF, once with methanol, once with DMF, once with methanol and twice with DMF, wherein the volume of each wash was 10 mL and the time length was 1 minute. The solvent was removed by vacuum suction. A small amount of dry resin was taken for ninhydrin detection. Both the resin and the solution were dark blue. Then, 462 mg MC-OSu was placed in a 50 mL centrifuge tube and about 10 mL DMF was added. By shaking, the solid was dissolved. Then, 0.24 mL DIEA was added to the resin. Stirring was performed to completely 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). Stirring was performed for 2-3 hours. After the reaction was completed, the reaction solution was removed by vacuum suction. The resin is washed with DMF twice, methanol once, DMF once, methanol once and DMF twice in sequence, wherein the volume of each washing is 10mL and the time length is 1 minute. Remove the solvent by vacuum suction. Take a small amount of dry resin and carry out ninhydrin detection. The resin is colorless and transparent, and the solution is slightly yellow, indicating that it is suitable for the next coupling step.

[0669] Step D-5

[0670] The resin was washed twice with 10 mL of methanol. Then, the solvent was completely removed by vacuum suction. The resin was poured out and weighed. A lysis buffer was prepared in a 250 mL conical flask, wherein: the ratio of TFE / DCM was 80% / 20%, and the volume was 7-8 times the weight of the peptide resin. The lysis buffer was added to the peptide resin and shaken thoroughly. The resin was completely immersed in the lysis buffer and lysed at room temperature for 2-3 hours. Next, the lysis buffer was filtered using a simple filter made of a syringe, and the resin was washed with 1-2 ml of DCM and discarded. Next, 150 mL of pre-cooled anhydrous ether was added to the lysis buffer, shaken thoroughly, and then left to stand for 20-30 minutes. Using a 50 mL centrifuge tube, the above system was centrifuged in a centrifuge at 3500 rpm for 3 minutes, and the supernatant was poured out and discarded. The solid was shaken with pre-cooled anhydrous ether, washed once under ultrasound, 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, followed by preparative purification to give 125 mg of the product as a white solid with a yield of 40%. MS m / z: [MH] - =641.5.

[0671] Preparation of Compound 22 (Step E)

[0672] 150 mg of raw material compound 21 and 55 mg of TSTU were weighed and added to a 10 mL single-necked round-bottom flask, anhydrous DMF (3 mL) was added and stirred for 20 minutes under a nitrogen atmosphere. Then, 18 mg of compound 12-1 and 20 μl of DIEA were added to the reaction system in sequence. Under a nitrogen atmosphere, stirred at room temperature for 2-8 hours. Samples were obtained and detected by HPLC to monitor the reaction. The raw material peak disappeared completely, and a new peak was detected.

[0673] The reaction system was subjected to preparative purification, and the target product was collected and lyophilized to obtain about 22 mg of the product in the form of a slightly yellow solid. MS m / z: [M+H] + =1081.0.

[0674] Preparation of Linker-Loader 3 (Step F, Step B)

[0675] Compound 22 (30 mg) was weighed and added to a 10 ml single-necked round-bottom flask, and purified water (2 ml) was added. Stir to dissolve. A DMF solution (2 ml) containing a linker-load intermediate 1 (19.5 mg) was added to the reaction system and stirred. After reacting overnight, the reaction was monitored using HPLC until all the raw materials had been converted into intermediates. An appropriate amount of Tris base solution or other solution that promotes the ring-opening reaction was directly added to the reaction mixture, and reacted for another 0.2-20 hours at 0-40 ° C. The reaction was monitored by HPLC until all the intermediates were consumed, and then quenched with acetic acid solution.

[0676] The reaction system was subjected to preparative purification, and the target product was collected and lyophilized to obtain about 25 mg of the linker-loaded substance 3 in the form of a slightly yellow solid. MS m / z: [(M+3H) / 3] + =1194.4.

[0677] Preparation of linker-carrier 4

[0678] The following linker-payload 4 can be prepared using a similar synthetic route and reagents as linker-payload 3. Linker-payload 4 is as follows:

[0679]

[0680] opSu is Example 2.3 Preparation of Linker-Loader 5

[0681]

[0682] Step 1: Preparation of intermediate compound b

[0683] Step 1.1 Preparation of NH2-Asp(OtBu)-Rink amide resin

[0684] 400 g of Rink amide resin was weighed and fully swollen with 2400 mL of DCM. 2400 mL of deprotection reagent was added to completely remove Fmoc, followed by washing with DMF and DCM several times at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0685] Weigh 88.87g Fmoc-Asp(OtBu)-OH and 29.19g HOBT and dissolve them in 2000mL DMF and 80mL DIC solution. After being placed in a -10°C ice bath for 0.5 hours, slowly add it to the reaction kettle containing the resin and stir the reaction at room temperature for 2-5 hours under nitrogen, then filter. The resin is washed with DMF and DCM successively, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0686] 2400 mL of deprotection reagent was added to completely remove Fmoc, followed by washing with DMF and DCM several times at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0687] Step 1.2 Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin

[0688] Weigh 131.64g Fmoc-PEG4-OH and 48.64g HOBT and dissolve them in 2000mL DMF and 80.0mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hours, slowly add it to the reaction kettle containing the resin, and stir the reaction at room temperature for 2-4 hours under nitrogen, then filter. The resin is washed with DMF and DCM successively, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0689] 2400 mL of deprotection reagent was added to completely remove Fmoc, followed by washing with DMF and DCM several times at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0690] Step 1.3 Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0691] Weigh 222.18g Fmoc-Asp(OtBu)-OH and 72.96g HOBT and dissolve them in 2000mL DMF and 80mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hour, slowly add it to the reaction kettle containing the resin, and stir the reaction at room temperature for 2-4 hours under nitrogen, then filter. The resin is washed with DMF and DCM successively, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0692] 2400 mL of deprotection reagent was added to completely remove Fmoc, followed by washing with DMF and DCM several times at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0693] Step 1.4 Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0694] Weigh 191.75g ​​Dde-Lys(Fmoc)-OH and 48.64g HOBT and dissolve them in 2000mL DMF and 80.0mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hour, slowly add it to the reaction kettle containing the resin, and under nitrogen, stir the reaction at room temperature for 2-4 hours, then filter. The resin is washed with DMF and DCM successively, and in the subsequent ninhydrin test, the resin shows colorless or light yellow.

[0695] 2400 mL of deprotection reagent was added to completely remove Fmoc, followed by washing with DMF and DCM several times at room temperature. In the subsequent ninhydrin test, the resin showed a blue color.

[0696] Step 1.5 Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0697] 170.84 g m-PEG12-CH2CH2COOH and 48.64 g HOBT were weighed and dissolved in 2000 mL DMF and 80.0 mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hours, they were slowly added to the reaction kettle containing the resin and stirred at room temperature for 2-4 hours under nitrogen, followed by filtration. The resin was washed with DMF and DCM successively, and in the subsequent ninhydrin test, the resin showed colorless or light yellow.

[0698] Step 1.6 Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0699] 2400 mL of the de-Dde reagent was added, and the reaction was stirred at room temperature for 0.5 hours under nitrogen, followed by filtration. After repeating this operation 3 times, the resin was washed successively with DMF and DCM, and in the subsequent ninhydrin test, the resin showed a blue color.

[0700] Step 1.7 Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0701] Weigh 111.08g Fmoc-Gly-Gly-Gly-OH and 48.64g HOBT and dissolve it in 2000mL DMF and 80.0mL DIC solution. After being placed in an ice bath at -10°C for 0.5 hour, it is slowly added to the reactor containing the resin, and under nitrogen, the reaction is stirred at room temperature for 2-4 hours, then filtered. The resin is washed with DMF and DCM successively, and in the subsequent ninhydrin test, the resin shows colorless or light yellow. The resin peptide is washed three times with absolute ethanol, filtered and waited for cutting.

[0702] Step 1.8 Preparation of intermediate compound b

[0703] 10000 mL of cleavage reagent (TFA:TIS:H2O=95:2.5:2.5) was added to a 10 L reactor and cooled to -10±2°C. The dried and weighed resin was added. The reactants were allowed to warm to room temperature and stirred under nitrogen for 2-3 hours. Afterwards, the resin was filtered and washed once with 100 mL of TFA. The filtrate was combined with the washing solution.

[0704] 40 L of pre-cooled (below -10°C) cold ether was added to the product solution. The mixture was stirred for 10 minutes, and then the precipitate was centrifuged. After centrifugation, the supernatant was discarded, the precipitate was collected and washed with cold ether, and the precipitate was centrifuged again (the speed of each centrifugation was set to 3600 rpm, the centrifugation time was 5 minutes, and the temperature of the centrifuge cavity was -5°C).

[0705] The precipitate was collected as crude compound b. The crude product was purified by preparative HPLC and lyophilized to give pure compound b.

[0706] Step 2: Preparation of intermediate compound a

[0707]

[0708] Step 2.1 Preparation of compound 2

[0709] Compound 1 (1 e.q.) and DMF (5 v / v) were added to a reaction flask, and the mixture was stirred and dissolved under nitrogen protection. After cooling the ice bath to 0-5°C, DIEA (3 e.q.) was added dropwise, and after the dropwise addition, the mixture was stirred at 5°C for 10 minutes. Next, benzyl bromide (1.3 eq) was added dropwise, and after the dropwise addition was completed, it was naturally warmed to room temperature of about 20°C and stirred for 16 hours.

[0710] The reaction solution was slowly poured into ice water, MTBE was added and stirred, and the solution was allowed to stand for separation. The aqueous phase was extracted 4 times with MTBE, the combined organic phase was washed with saturated brine, then the organic phase was dried over anhydrous sodium sulfate, and concentrated under vacuum to give a crude yellow oil, which was applied to the column in a wet method. Elution by PE / EA=6:1 gave a light yellow oil, and the yield was 100%.

[0711] Step 2.2 Preparation of compound 4

[0712] Under nitrogen protection, intermediate 2 (2.0eq), compound 3 (1e.q.) and THF (10v / v) were added to a reaction flask, and stirred to dissolve, TsOH (0.1eq) was weighed and added to the reactant, and the reaction was maintained at 20-22°C for 4 hours. The reaction solution was slowly poured into ice water, extracted 3 times with EA, and the combined organic phase was sequentially washed with saturated sodium bicarbonate aqueous solution, water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and dried under vacuum to obtain a crude product. The silica gel sample was mixed and passed through the column, eluted with PE / EA=1:1, the product was collected, and concentrated to obtain a white solid with a yield of 40%.

[0713] Step 2.3 Preparation of compound 7

[0714] Under nitrogen protection, compound 4 and DMAc (10v / v) were added to a reaction flask and stirred to dissolve. The reactant was cooled to 14-18°C, DBU (0.5eq) was added dropwise and the reaction was stirred at this temperature for 1.5 hours, and the completion of the raw material reaction was monitored by TLC. The reactant was cooled to 0-5°C, PPTS (0.5eq), EDCI (1e.q.), HOBT (1e.q.) and compound 6 (0.85eq) were added and reacted at 0-10°C for 3-4 hours, and the reaction was monitored by LCMS.

[0715] The reaction solution is added to ice water, 2-methyltetrahydrofuran is added and extracted once, and the aqueous phase is extracted twice with 2-methyltetrahydrofuran. The organic phase is merged, 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 is collected by DCM / MeOH wash-out, and dried under vacuum to obtain a white solid in a yield of 78%.

[0716] Step 2.4 Preparation of compound 10

[0717] Under nitrogen protection, intermediate 7 and DMAc (10v / v) were added to the reaction flask and stirred to dissolve. The reactant was cooled to 14-18°C, DBU (0.5eq) was added dropwise, and the reaction was stirred for 1.5 hours at this temperature, and the completion of the reaction was monitored by TLC. The reactant was cooled to 0-5°C, PPTS (0.5eq), EDCI (1e.q.), HOBT (1e.q.) and compound 9 (0.85eq) were added and reacted at 0-10°C for 3-4 hours, and the reaction was monitored by LCMS.

[0718] The reaction solution is added to ice water, 2-methyltetrahydrofuran is added and extracted once, and the aqueous phase is extracted twice with 2-methyltetrahydrofuran. The organic phase is merged, washed with 0.5M hydrochloric acid, saturated NaHCO3 aqueous solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, evaporated to dryness, mixed with silica gel, and purified by column. The product is collected by DCM / MeOH wash-out, and dried under vacuum to obtain a white solid in a yield of 50%.

[0719] Step 2.5 Preparation of compound a

[0720] Under nitrogen protection, intermediate 10 was dissolved in DCM (15 v / v), DBU (0.5 eq) was added dropwise at 20 ° C, and the reaction was stirred for 5 hours at 18-22 ° C. Complete reaction was monitored by LCMS. The reaction solution was diluted with DCM, column purified by wet method, and the product was collected by DCM: MeOH elution to obtain a white solid with a yield of 82%.

[0721] Step 3: Preparation of intermediate compound c

[0722]

[0723] Compound b (400 mg, 0.245 mmol) and compound a (377 mg, 0.539 mmol) were dissolved in DMF (6 ml), and then DIPEA (159 mg, 1.23 mmol) and HATU (233 mg, 0.613 mmol) were added to the reaction solution, and the reaction was stirred at room temperature for 2 hours. After compound b disappeared, it was purified by preparative HPLC, and the prepared solution was lyophilized to obtain 380 mg of product with a yield of 52%. 142 H 207 O 49 N 21 [(M+3H) / 3] + Calculated value: 997.8, found value: 875.9 (mass of cleavage).

[0724] Step 4: Preparation of intermediate compound d

[0725]

[0726] Compound c (380 mg, 0.245 mmol) was dissolved in purified water (80 ml), and palladium hydroxide (38 mg) was added. The system was replaced with hydrogen three times and stirred at room temperature for 1.5 hours. During this period, the reaction progress was monitored and the reaction was stopped immediately after the raw material disappeared to prevent the increase of the de-Fmoc product. The reaction solution was filtered and purified by preparative HPLC to obtain 270 mg of product with a yield of 76%. 128 H 195 O 49 N 21 [(M+3H) / 3] + Calculated value: 937.8, found value: 875.9 (mass of cleavage).

[0727] Step 5: Preparation of intermediate compound e

[0728]

[0729] 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, and the collected eluents were combined and lyophilized to obtain 235 mg of the product with a yield of 66%. 174 H 229 O 55 Cl2F2N 27[(M+3H) / 3] + Calculated value: 1229.2, experimental value: 1229.3.

[0730] Step 6: Preparation of Linker-Loader 5

[0731]

[0732] Compound e (210 mg, 0.057 mmol) was dissolved in DMF (5 ml), followed by the addition of diethylamine (0.5 ml) and the reaction was allowed to react at room temperature for 15 minutes, and the reaction endpoint was monitored by HPLC. After the reaction was completed, it was adjusted to neutrality with 10% TFA aqueous solution under ice bath, and the reaction was purified by preparative HPLC to obtain 145 mg of product after freeze drying, with a yield of 73%. 159 H 219 O 53 Cl2F2N 27 [(M+3H) / 3] + Calculated value: 1155.2, experimental value: 1155.3.

[0733] Example 3 Preparation of Targeting Molecule-Drug Conjugates

[0734] 3.1 Preparation of DS1062a analogs and Trodelvy

[0735] DS1062a analogs (DS1062a and DS-1062 * ) is prepared according to the method described in patent US20160297890A or produced by WuXi Biologics.

[0736] Trodelvy is commercially available.

[0737] 3.2 Preparation of ADC-1

[0738] 3.2.1 GQhRS7 treatment

[0739] Treat GQhRS7 by ultrafiltration, dialysis or desalting column. Replace the storage solution with ligase buffer.

[0740] 3.2.2 Enzyme-catalyzed ADC-1 conjugation

[0741] ADC-1 is prepared by coupling GQhRS7 with linker-load 1 under the catalysis of wild-type sortase A or a mutant ligase optimized or engineered based on the sortase A. In the ligase buffer, the modified antibody and linker-load are fully mixed at a molar ratio of 1:1 to 1:100 and added to the solid phase coupling system. The solid phase coupling system comprises a ligase immobilized on a matrix of the solid phase coupling system. The immobilized ligase catalyzes the coupling reaction of the antibody GQhRS7 with the linker-load 1. The coupling reaction is carried out at 4-40°C for 0.5-20 hours. After the reaction is completed, the reaction mixture is ultrafiltered or dialyzed to remove unreacted intermediates to obtain ADC-1. ADC-1 is stored in a pH 5.0 buffer containing 20mM citric acid and 200mM NaCl at 4°C or -80°C.

[0742] 3.2.3 HIC-HPLC detection and analysis of ADC-1

[0743] The DAR (drug to antibody ratio) distribution of ADC-1 was analyzed by HIC-HPLC. Antibody GQhRS7 was less than 5% in the absence of cytotoxins; and the conjugated product mainly contained ADC-1, and the DAR was 3.5.

[0744] 3.2.4 SEC-HPLC detection and analysis of ADC-1

[0745] The degree of high molecular weight aggregation of ADC-1 was analyzed by SEC-HPLC. The results showed that no high molecular weight aggregates were detected in ADC-1, indicating that the coupling reaction conditions were mild and did not cause damage to the antibody structure.

[0746] 3.3 The linker-load intermediate is conjugated to the antibody in a site-specific manner by a ligase to form an ADC. The conjugation reaction method can be found in WO2015165413A1. The obtained ADC is listed in the following table:

[0747] Name of ADC Linker-Payload Antibody ADC-1 Linker-Load 1 QUR ADC-2 Linker-Load 3 QUR ADC-3 Linker-Loader 4 QUR ADC-4 Linker-Load 5 QUR ADC-5 Linker-Load 5 GQAb13 ADC-6 Linker-Load 5 GQAb16 ADC-7 Linker-Patent 2 QUR

[0748] 3.4 Binding activity

[0749] Human Trop 2ECD at a concentration of 0.5 μg / mL was coated on a 96-well plate and kept overnight at 4°C. Then, at room temperature, the plate was blocked with 3% BSA-PBST for 1 hour. After washing with PBST (0.05% Tween), a series of test samples including ADC-2 (i.e., ADC2), Trodelvy and GQhRS7 at different concentrations were added to the 96-well plate, followed by incubation 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 for another 60 minutes at room temperature. After washing, the plate was treated with TMB solution (Sigma, T0440) as an HRP substrate, and the reaction was stopped with 1M H2SO4. The absorbance of each well was detected at a wavelength of 450nm.

[0750] The results of this analysis are shown in Figure 1.2 The results showed that ADC-2 had similar binding affinity to GQhRS7 and Trodelvy.

[0751] 3.5 Internalization activity

[0752] NCI-N87 with good vitality was treated with trypsin, collected, and suspended in cold FACS buffer (DPBS + 2% FBS). The cells were incubated on ice for one hour with a test drug solution at a final concentration of 50 μg / ml. The antibody-cell mixture was washed twice with cold FACS buffer to remove excess antibody. The antibody-bound cells were fluorescently labeled by mixing with a 500-fold diluted ice-cold anti-human IgG-Fc-AF647 secondary antibody solution for 30 minutes. After fluorescent labeling, the antibody-cell mixture was washed twice again. The cells were incubated at 37 ° C for 10 minutes, 30 minutes, 60 minutes, 90 minutes, 150 minutes and 210 minutes to internalize the test drug. The cells were suspended with quenching buffer (150mM NaCl + 100mM glycine, pH = 2.0 to 2.5) to dissociate the antibodies bound on the cell surface. After acidification, the cells were washed twice with cold FACS buffer and analyzed by flow cytometry in the APC channel.

[0753] Substitute the MFI data into the following formula and analyze the results using a one-phase exponential correlation function in Prism8. Internalization amount: A = MFI(I) - MFI(MIN). Internalization ratio:

[0754] R=[MFI(I)-MFI(MIN)] / [MFI(MAX)-MFI(Blank)]×100%

[0755] like Figure 1.3As shown in , ADC2 showed similar internalization activity to DS1062, Trodelvy, and GQhRS7 on NCI-N87.

[0756] Effect Example 1 Bystander Killing Effect of the Conjugate in BxPC-3 / HepG2

[0757] Effect Example 1.1 Bystander Killing of HepG2 by ADC-1 in BxPC-3 / HepG2 Co-culture Assay

[0758] The cell concentration of Trop2-positive BxPC-3 cells and Trop2-negative HepG2 cells was adjusted to 1×10 6 Cells / ml, and 200 μL per well (cell volume BxPC-3:HepG2=4:1) were inoculated into a six-well plate and supplemented with 2.8 mL of 45% RPMI-1640+45% DMEM+10% FBS culture medium. In a cell incubator, the cells were cultured overnight at 37°C and 5% CO2. 3 mL of 20 nM ADC-1 and DS1062a were added to the cells, respectively, and cultured overnight (the final drug concentration was 10 nM per well). The negative control group was set as follows: 3 mL of 45% RPMI-1640+45% DMEM+10% FBS culture medium was added to each well. After treatment, the cells were moved to an incubator and cultured for 96 hours. After incubation, the cells were digested and washed once with 1×PBS, then transferred to a flow tube and centrifuged at 2000 rpm for 3 minutes. Then, the supernatant was discarded, and the cell amount and cell viability were detected. A certain amount of cells were washed with 1×PBS, the supernatant was discarded after centrifugation, 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 washed with 1×PBS, the supernatant was discarded after centrifugation, 200 μL of 5 μg / mL human IgG Fc antibody was added, and after mixing, the resulting cells were further incubated at 4°C for 30 minutes. Finally, the cells were washed with 1×PBS, the supernatant was discarded after centrifugation, and the cells were resuspended in PBS, detected by flow cytometry and analyzed by FlowJo software. The results are shown in Table 4.

[0759] Table 4 Test results of bystander killing of ADC-1 and DS1062a

[0760]

[0761] Effect Example 1.2 Bystander killing effect of ADC-2 against HepG2 in BxPC-3 / HepG2 co-culture assay

[0762] The cell concentration of Trop2-positive BxPC-3 cells and Trop2-negative HepG2 cells was adjusted to 1×10 6 Cells / ml, and 300 μL per well (cell volume BxPC-3:HepG2=2:1) ​​were inoculated into a six-well plate, and supplemented with 2.7 mL of 45% RPMI-1640+45% DMEM+10% FBS culture medium. In a cell incubator, the cells were cultured overnight at 37°C, 5% CO2. 3 mL of 20 nM ADC-2 and DS1062a were added to the cells, respectively, and cultured overnight (the final drug concentration was 10 nM per well). And the next process was to use the same method as above to evaluate the bystander killing effect of ADC-2 and DS1062a. The results are shown in Table 5.

[0763] Table 5. Test results of bystander killing for ADC-2 and DS1062a

[0764]

[0765] Effect Example 1.3 Bystander killing effect of ADC-6 against HepG2 in BxPC-3 / HepG2 co-culture assay

[0766] The bystander killing effect of control (dabituzumab), DS1062a, ADC-1, ADC-2, ADC-4 and ADC-6 was evaluated using the same method as in efficacy example 1.2. The results are shown in Table 6.

[0767] Table 6 Test results of bystander killing injury of ADC-6

[0768]

[0769] in conclusion

[0770] The experimental results of Action Example 1.1 showed that both ADC-1 and DS1062a analogs had bystander killing effects, and there was no significant difference in their efficacy.

[0771] The experimental results of Action Example 1.2 showed that the bystander killing effect of ADC-2 was superior to that of DS1062a.

[0772] The experimental results of Action Example 1.3 show that ADC-1, ADC-2, ADC-4, ADC-6 and DS1062a analogs all have bystander killing effects. The bystander killing effects of ADC-2 and ADC-6 are better than the bystander killing effect of DS1062a. The bystander killing effect of ADC-6 is significantly better than the bystander killing effect of ADC-4.

[0773] Effect Example 2: Effect of TROP2-targeted conjugates on cell proliferation

[0774] Effect Example 2.1 Inhibitory Effects of ADC-7 and ADC-1 on Tumor Cell Proliferation

[0775] Effect Example 2.1.1 Inhibitory effect of ADC-7 on proliferation of human pharyngeal squamous cell carcinoma FaDu

[0776] Two thousand human pharyngeal squamous cell carcinoma FaDu with high TROP2 expression were seeded in 96-well plates with 100 μL of culture medium per well and incubated overnight in a cell incubator at 37°C and 5% CO2. 100 μL of ADC-7 and DS1062a of different concentrations (200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, 0.000512 nM and 0.0001024 nM) 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 complete culture medium containing FaDu cells was added to each well. After the administration was completed, the cells were moved to an incubator and incubated for 96 hours. The 96-well plate was removed from the 37°C cell incubator and equilibrated to room temperature for 30 minutes. After discarding the culture medium, 100 μL of DMEM and 50 μL of CellTiter Glo reagent were added to each well, and the cells were shaken at 200 rpm for 15 minutes in the dark, and the luminescent signal, which reflects cell viability, was detected by 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.

[0777] Table 7 Inhibitory effects of ADC-7 and DS1062a on proliferation of pharyngeal squamous cell carcinoma FaDu

[0778]

[0779] Effect Example 2.1.2 Inhibitory effect of ADC-1 on proliferation of human pancreatic cancer cells BxPC-3

[0780] The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human pancreatic cancer cells BxPC-3 (100 μL per well containing 2000 cells), and the inhibitory effect of ADC-1 was evaluated using the same method. 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.

[0781] Table 8 Inhibitory effects of ADC-1 and DS1062a on proliferation of human pancreatic cancer cells BxPC-3

[0782]

[0783] Effect Example 2.1.3 Inhibitory effect of ADC-1 on proliferation of human breast cancer cells MDA-MB-468

[0784] The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human breast cancer cells MDA-MB-468 (100 μL per well containing 4000 cells), and the same method was used to evaluate the inhibitory effect of ADC-1. The inhibitory effect of the test drug on the proliferation of human breast cancer cells MDA-MB-468 is shown in Figure 4 and Table 9.

[0785] Table 9 Inhibitory effects of ADC-1 and DS1062a on proliferation of human breast cancer cells MDA-MB-468

[0786]

[0787] Effect Example 2.1.4 Inhibitory effect of ADC-7 on proliferation of human gastric cancer cell NCI-N87

[0788] The human pharyngeal squamous cell carcinoma FaDu in Example 2.1.1 was replaced with human gastric cancer cell NCI-N87 (100 μL per well containing 5000 cells), and the inhibitory effect of ADC-7 was evaluated using the same method. The inhibitory effect of the test drug on the proliferation of human gastric cancer cell NCI-N87 is shown in Figure 5 and Table 10.

[0789] Table 10 Inhibitory effects of ADC-7 and DS1062a on proliferation of gastric cancer cell NCI-N87

[0790]

[0791] Effect Example 2.2 Inhibitory Effects of ADC-2 and ADC-3 on Tumor Cell Proliferation

[0792] Effect Example 2.2.1 Inhibitory Effects of ADC-2 and ADC-3 on Proliferation of Human Pharyngeal Squamous Cell Carcinoma FaDu

[0793] The test drugs ADC-1 and DS1062a in Example 2.1.1 were replaced with ADC-2, ADC-3 and ADC-1, and the inhibitory effect was evaluated using the same method. The inhibitory effect of the test drugs on the proliferation of human pharyngeal squamous cell carcinoma FaDu is shown in Figure 6 and Table 11.

[0794] Table 11 Inhibitory effects of ADC-2, ADC-3 and ADC-1 on proliferation of pharyngeal squamous cell carcinoma FaDu

[0795]

[0796] Effect Example 2.2.2 Inhibitory Effects of ADC-2 and ADC-3 on Proliferation of Human Pancreatic Cancer Cells BxPC-3

[0797] The human pharyngeal squamous cell carcinoma FaDu in Example 2.2.1 was replaced with human pancreatic cancer cells BxPC-3, and the same method was used to evaluate the inhibitory effect of ADC-2. The inhibitory effect of the test drug on the proliferation of human pancreatic cancer cells BxPC-3 is shown in Figure 7.1 and Table 12.

[0798] Table 12 Inhibitory effects of ADC-2, ADC-3 and ADC-1 on proliferation of human pancreatic cancer cells BxPC-3

[0799]

[0800] Effect Example 2.3 Inhibitory Effects of ADC-2 and DS-1062a on Proliferation of BxPC-3, FaDu and NCI-N87

[0801] Using Trop2-positive cancer cells BxPC-3 ( Figure 7.2 )、FaDu( Figure 7.3 ) and NCI-N87( Figure 7.4 ) performed cytotoxicity assays to analyze the effects of the conjugates on tumor cell proliferation. Test drugs included conjugates ADC2, DS1062a, and GQhRS7. Briefly, 3000 to 5000 cells were plated in 96-well plates and allowed to attach overnight. Cells were treated with various concentrations of the designated drugs for 168 hours. Cell viability was examined by luminescence cell viability assay, and the percentage of cell viability was calculated.

[0802] In Trop2-positive BxPC-3, FaDu, and NCI-N87, ADC2 exhibited more potent cytotoxicity than DS1062a. 50 Values ​​are smaller than those of the DS1062a (see table below).

[0803]

[0804] in conclusion

[0805] 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.

[0806] The results of effect example 2.1.2 showed that both ADC-1 and DS1062a could inhibit the proliferation of BxPC-3 cells.

[0807] The results of effect example 2.1.3 showed that both ADC-1 and DS1062a could inhibit the proliferation of MDA-MB-468 cells.

[0808] The results of effect example 2.1.4 showed that both ADC-7 and DS1062a could inhibit the proliferation of NCI-N87 cells.

[0809] The results of effect example 2.2.1 showed that ADC-2, ADC-3 and ADC-1 could inhibit the proliferation of FaDu cells.

[0810] The results of effect example 2.2.2 showed that ADC-2, ADC-3 and ADC-1 could inhibit the proliferation of BxPC-3 cells, and the inhibitory effect of ADC-2 was slightly better than that of ADC-3 and ADC-1.

[0811] The results of effect example 2.3 showed that ADC-2 could inhibit the proliferation of BxPC-3 cells, FaDu cells and NCI-N87 cells, and the inhibitory effect of ADC-2 was superior to that of DS1062a.

[0812] Effect Example 3 In vivo efficacy evaluation test

[0813] Effect Example 3.1 In vivo efficacy evaluation of ADC-1

[0814] Effect Example 3.1.1 Evaluation of the in vivo efficacy of ADC-1 against BxPC-3 cells

[0815] I. Collect BxPC-3 cells in the logarithmic growth phase and adjust the cell density to 10×10 6 0.2 mL of the prepared BxPC-3 cell suspension was subcutaneously injected into the right scapula of 6-8 week old SPF female BALB / c nude mice.

[0816] II. Measure the tumor diameter with a vernier caliper and calculate the tumor volume according to the following formula: V = 0.5a × b 2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor). Six days after cell inoculation, when the average tumor volume was approximately 151 mm 3At 1:10 pm, the animals were randomly divided 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. Each group of animals was administered by tail vein injection, and the control group was given an equal volume of vehicle. Within 35 days after administration, the tumor volume of each group of animals was measured twice a week, and the tumor volume of each group of animals was compared on the 35th day. The tumor volume was used to calculate the T / C and TGI values. The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV : RTV of treatment group; C RTV : RTV of the vehicle control group). Relative tumor volume (RTV) was calculated based on tumor measurement results, and the calculation formula is: RTV = V t / V0, where V0 is the mean tumor volume measured at the time of grouping (i.e., D0), V t is the average tumor volume at one measurement, and T RTV and C RTV The data of the same day were used. Calculation of TGI (%): TGI (%) = [1-(average tumor volume at the end of treatment group administration-average tumor volume at the beginning of treatment group administration) / (average tumor volume at the end of vehicle control group treatment-average tumor volume at the beginning of vehicle control group treatment)] × 100%.

[0817] III. After 35 days of medication, 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 847mm 3 The results are shown in Figure 8 and Table 13.

[0818] Table 13 Inhibitory effect of ADC-1 on BxPC-3 mouse xenograft tumors

[0819]

[0820] in conclusion

[0821] According to the results of effect example 3.1.1, compared with the control group, both the ADC-1 3 mg / kg group and the DS1062a 3 mg / kg group could significantly inhibit tumor growth.

[0822] Effect Example 3.1.2 Evaluation of the in vivo efficacy of ADC-1 against NCI-N87 gastric cancer cells

[0823] The BxPC-3 cells in Example 3.1.1 were replaced with NCI-N87 gastric cancer cells, and 7 days after cell inoculation, when the average tumor volume was about 227 mm 3 At 1 , the animals were randomly divided into a vehicle control group, an IMMU-132 (Trodelvy) 3 mg / kg group, a DS1062a 3 mg / kg group, and an ADC-1 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-1 was evaluated using a method similar to that of Effect Example 3.1.1, and the results are shown in Fig. 9 and Table 14.

[0824] Table 14 Inhibitory effect of ADC-1 on NCI-N87 mouse xenograft tumors

[0825]

[0826] in conclusion

[0827] After 32 days of treatment, the average tumor volumes 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 658mm 3 , they can all significantly inhibit tumor growth.

[0828] Effect Example 3.1.3 Evaluation of the in vivo efficacy of ADC-1 against human breast cancer BR-05-0028

[0829] The human breast cancer BR-05-0028 model (IHC 3+) was derived from a tumor sample removed during clinical surgery. The P0 generation tumor sample was inoculated into nude mice, and the tumor tissue used in this example was the P5 generation. 3 Tumor tissues of 1.5-2.0 mm in size were subcutaneously inoculated into the right back of 6-8 week-old SPF female BALB / c nude mice. 28 days after inoculation of tumor tissues, when the average tumor volume was about 171 mm 3 At 1 , 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 Fig.10 and Table 15.

[0830] Table 15 Inhibitory effect of ADC-1 on BR-05-0028 mouse xenograft tumors

[0831]

[0832] in conclusion

[0833] After 21 days of medication, the IMMU-132 5 mg / kg group (T / C=0.35%, TGI=111.76%, p=0.037), the DS1062a 5 mg / kg group (T / C=0.48%, TGI=111.60%, p=0.037), the ADC-1 5 mg / kg group (T / C=0.23%, TGI=111.88%, p=0.037) and the ADC-1 10 mg / kg group (T / C=0.23%, TGI=111.88%, p=0.037) were all able to significantly inhibit tumor growth.

[0834] Effect Example 3.2 Evaluation of the in vivo efficacy of ADC-2 and ADC-3

[0835] Effect Example 3.2.1 Evaluation of the in vivo efficacy of ADC-2 and ADC-3 against BxPC-3 cells

[0836] Six days after cell inoculation, when the average tumor volume was approximately 151 mm 3 At 1 , 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 Fig.11 and Table 16.

[0837] Table 16 Inhibitory effects of ADC-2 and ADC-3 on BxPC-3 mouse xenograft tumors

[0838]

[0839] in conclusion

[0840] After 28 days of medication, 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 540mm 3 , which indicates that the ADC-2 3 mg / kg group can significantly inhibit tumor growth.

[0841] Effect Example 3.2.2 Evaluation of the in vivo efficacy of ADC-2 and ADC-3 against NCI-N87 gastric cancer cells

[0842] 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 mm 3 At 1 , 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 was evaluated using a method similar to that of Example 3.2.1, and the results are shown in Fig.12 and Table 17.

[0843] Table 17 Inhibitory effect of ADC-2 on NCI-N87 mouse xenograft tumors

[0844]

[0845] in conclusion

[0846] After 28 days of medication, the average tumor volumes of the ADC-2 3 mg / kg group (T / C=14.08%, TGI=122.25%, p<0.001), ADC-3 3 mg / kg group (T / C=33.43%, TGI=94.72%, p<0.001), ADC-1 3 mg / kg group (T / C=46.15%, TGI=76.62%, p<0.001) and DS1062a 3 mg / kg group (T / C=40.30%, TGI=84.96%, p<0.001) were 89, 212, 293 and 255 mm, respectively. 3 , indicating that all tested drugs could significantly inhibit NCI-N87 tumor growth.

[0847] Effect Example 3.2.3 Evaluation of the in vivo efficacy of ADC-2 and ADC-3 against human pharyngeal squamous cell carcinoma FaDu

[0848] The BxPC-3 cells in Example 3.2.1 were replaced with human pharyngeal squamous cell carcinoma FaDu, and 11 days after cell inoculation, when the average tumor volume was about 123 mm 3 At 1 , the animals were randomly divided into vehicle control, DS1062a 3 mg / kg group, ADC-1 3 mg / kg group, ADC-2 3 mg / kg group and ADC-2 3 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-2 was evaluated using a method similar to that of Example 3.2.1, and the results are shown in Figure 13.1 and Table 18.

[0849] Table 18 Inhibitory effect of ADC-2 on FaDu mouse xenograft tumors

[0850]

[0851] in conclusion

[0852] After 28 days of medication, the average tumor volumes of the ADC-2 3 mg / kg group (T / C=0.00%, TGI=107.90%, p=0.003), ADC-3 3 mg / kg group (T / C=0.25%, TGI=107.63%, p=0.003), ADC-1 3 mg / kg group (T / C=1.23%, TGI=106.59%, p=0.003) and DS1062a 3 mg / kg group (T / C=1.68%, TGI=106.11%, p=0.003) were 0, 4, 21 and 28 mm, respectively. 3 , indicating that all tested drugs could significantly inhibit FaDu tumor growth. In addition, at the end of the experiment, 6 mice in the ADC-2 3mg / kg group and 3 mice in the ADC-3 3mg / kg group showed complete tumor regression.

[0853] Effect Example 3.2.4 Evaluation of the in vivo efficacy of ADC-2 against MDA-MB-468

[0854] 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 and 0% CO2 in an air environment. Cells growing in the exponential growth phase were collected and counted for tumor inoculation. For in vivo antitumor efficacy studies, 10×10 6 MDA-MB-468 human breast cancer cells (Trop2 positive) were subcutaneously inoculated into the right flank of BALB / c nude mice with 0.2 mL of Matrigel-containing PBS (1:1). After 24 days, when the tumor volume reached an average of 187 mm 3 At 1:10 pm, tumor-bearing mice were grouped and intravenously administered 0.5 mg / kg, 1.5 mg / kg and 4.5 mg / kg of ADC-2, 4.5 mg / kg of Trodelvy and 4.5 mg / kg of DS1062a. Tumor volume was measured with a caliper twice a week. T / C and TGI values ​​were calculated using tumor volume.

[0855] in conclusion

[0856] After 35 days of medication, the ADC-2 4.5 mg / kg group was: T / C = 0.00%, TGI = 137.93%; the DS1062a 4.5 mg / kg group was T / C = 1.35%, TGI = 136.06%; the Trodelvy 4.5 mg / kg group was T / C = 78.73%. The results are shown in Figure 13.2 ADC-2 showed significantly better efficacy than Trodelvy and slightly better efficacy than DS1062a.

[0857] Inhibitory effect of ADC-2 on xenograft tumors in MDA-MB-468 mice

[0858]

[0859] Effect Example 3.3 Evaluation of the in vivo efficacy of ADC-5 and ADC-6

[0860] Effect Example 3.3.1 Evaluation of the in vivo efficacy of ADC-5 and ADC-6 against BxPC-3 pancreatic cancer cells

[0861] Six days after cell inoculation, when the average tumor volume was approximately 159 mm 3 At 1 , 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 Example 3.1.1, and the results are shown in Fig.14 and Table 19.

[0862] Table 19 Inhibitory effects of ADC-5 and ADC-6 on BxPC-3 mouse xenograft tumors

[0863]

[0864] in conclusion

[0865] After 42 days of medication, the average tumor volumes of the ADC-5 3 mg / kg group (T / C=0.75%, TGI=116.20%, p=0.003), ADC-6 3 mg / kg group (T / C=2.48%, TGI=114.17%, p=0.003) and DS1062a 3 mg / kg group (T / C=27.69%, TGI=84.60%, p=0.007) were 8 mm 3 、27mm 3 and 302mm 3 , indicating that all tested drugs could significantly inhibit tumor growth; the inhibitory effects of ADC-5 and ADC-6 were significantly better than that of DS1062a.

[0866] Effect Example 3.3.2 Evaluation of the in vivo efficacy of ADC-5 and ADC-6 against NCI-N87 gastric cancer cells

[0867] The BxPC-3 pancreatic cancer cells in Example 3.3.1 were replaced with NCI-N87 gastric cancer cells, and 6 days after cell inoculation, when the average tumor volume was about 196 mm 3 At 1 , 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 Example 3.3.1, and the results are shown in Fig.15 and Table 20.

[0868] Table 20 Inhibitory effects of ADC-5 and ADC-6 on NCI-N87 mouse xenograft tumors

[0869]

[0870] in conclusion

[0871] After 42 days of medication, the average tumor volumes of the ADC-5 3 mg / kg group (T / C=10.88%, TGI=106.06%, p<0.001), ADC-6 3 mg / kg group (T / C=3.89%, TGI=114.36%, p<0.001) and DS1062a 3 mg / kg group (T / C=35.67%, TGI=76.58%, p<0.001) were 133 mm 3 、48mm 3 and 437mm 3 , indicating that all tested drugs could significantly inhibit tumor growth; the inhibitory effects of ADC-5 and ADC-6 were significantly better than that of DS1062a.

[0872] Effect Example 3.3.3 Evaluation of the in vivo efficacy of ADC-5 and ADC-6 against human pharyngeal squamous cell carcinoma FaDu

[0873] The BxPC-3 pancreatic cancer cells in Example 3.3.1 were replaced with human pharyngeal squamous cell carcinoma FaDu, and 10 days after cell inoculation, when the average tumor volume was about 119 mm 3 At 1 , the animals were randomly divided into a vehicle control group, an ADC-5 2 mg / kg group, an ADC-6 2 mg / kg group, and a DS1062a 2 mg / kg group, with 6 animals in each group. The in vivo efficacy of ADC-5 and ADC-6 was evaluated using a method similar to that of Example 3.3.1, and the results are shown in Fig.16 and Table 21.

[0874] Table 21 Inhibitory effects of ADC-5 and ADC-6 on FaDu mouse xenograft tumors

[0875]

[0876] in conclusion

[0877] After 31 days of medication, the average tumor volumes of the ADC-5 2 mg / kg group (T / C=11.34%, TGI=94.56%, p=0.004), ADC-6 2 mg / kg group (T / C=0.05%, TGI=106.60%, p=0.004) and DS1062a 2 mg / kg group (T / C=15.47%, TGI=90.15%, p=0.005) were 217 mm 3 , 1mm 3 and 296mm 3 , indicating that all tested drugs could significantly inhibit tumor growth; the inhibitory effect of ADC-6 was significantly better than that of DS1062a.

[0878] Effect Example 4 Serum stability of ADC-1

[0879] An appropriate amount of Trop 2 was covalently coupled to CNBr-activated agarose microspheres to form an immobilized antigen. After blocking, a certain proportion of the stabilized sample pre-incubated with plasma was added and incubated under shaking. ADC-1 and DS1062a (ADC drug) in the matrix were captured by fixed antigen-specificity to form a solid phase antigen / antibody complex, and unbound substances were removed by washing. After incubation, N-glycosidase was used to remove the coupled sugar chain in the Fc region of the ADC drug antibody, followed by formic acid elution to recover the ADC, and DAR was detected by LC-MS. The results are shown in Fig.17 middle.

[0880] in conclusion

[0881] The results showed that the serum stability of ADC-1 was significantly better than that of DS1062a.

Claims

1. An anti-TROP2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (V H ) and light chain variable region (V L ),in The V H Include: (i) a HCDR1 comprising 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) a HCDR2 comprising 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) comprising the amino acid sequence X 10 HCDR3 of GFGSSYWYFDV (SEQ ID NO: 3), wherein X 10 is G or S; and / or The V L Include: (i) LCDR1 comprising the amino acid sequence KASQDVSIAVA (SEQ ID NO: 13) or KASQDVSTAVA (SEQ ID NO: 14); (ii) LCDR2 comprising the amino acid sequence SASYRYT (SEQ ID NO: 15); and (iii) LCDR3 comprising the amino acid sequence QQHYITPLT (SEQ ID NO: 16); Preferably, The V H Include: (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and / or The V L Include: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (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; or The V H Include: (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 5, (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and / or The V L Include: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, (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; or The V H Include: (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, (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 The V L Include: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (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; or The V H Include: (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, (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 The V L Include: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (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; or The V H Include: (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and (iii) HCDR3 comprising the amino acid sequence of SEQ ID NO: 11; and / or The V L Include: (i) LCDR1 comprising the amino acid sequence of SEQ ID NO: 13, (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.

2. The antibody or antigen-binding fragment according to claim 1, wherein the V H comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NOs: 21 to 25; and / or The V L comprising 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; and / or The antibody or antigen-binding fragment comprises: a heavy chain constant domain (CH), wherein the CH comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 28; and / or The antibody or antigen-binding fragment comprises: a light chain constant domain, wherein the light chain constant domain comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:

29.

3. The antibody or antigen-binding fragment of claim 1, comprising: a heavy chain comprising an amino acid sequence having at least about 90% sequence identity with 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 with the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO:

35.

4. The antibody or antigen-binding fragment according to claim 3, wherein the C-terminal modification of the heavy chain and / or the C-terminal modification of the light chain are included, thereby the antibody, Sp and the recognition sequence of the ligase donor substrate are sequentially connected; Sp is a spacer sequence selected from GA, GGGGS (SEQ ID NO: 42), GGGGSGGGGS (SEQ ID NO: 43) and GGGGSGGGGSGGGGS (SEQ ID NO: 44); the recognition sequence of the ligase donor substrate is LPXTGJ (SEQ ID NO: 49), wherein X can be any single natural or unnatural amino acid; J is absent or is an amino acid fragment comprising 1-10 amino acids.

5. A polynucleotide or vector comprising a nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of claims 1 to 4. A host cell comprising the polynucleotide or the vector according to claim 5 .

7. A conjugate having a structure of formula (III): in Q is hydrogen, -C2H4-(PEG) t -(CO)NH2 or LKb-P; M is hydrogen or LKa-LKb―P; in Each LKa is independently selected from opSu is or a mixture thereof; Each LKb is independently L 2 ―L 1 ―B; Each B is independently absent, or is a combination of 1) and 2): 1) a self-removing spacer Sp1; and 2) a chemical bond, or a combination of one or two or more of the following divalent groups: -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 is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-; U is absent, or is O, S or NH, preferably O or S; Provided that Q and M are not simultaneously hydrogen; P is a load, which is connected to the B part or L of formula (III) 1 part; Each L 1 is independently a cleavable sequence 1 comprising an amino acid sequence capable of being cleaved by an enzyme, and the cleavable sequence 1 comprises 1-10 amino acids; Each L 2 independently a chemical bond; or C 2-20 Alkylene, wherein one or more -CH2- structures in the 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 wherein the cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or substituted by at least one substituent selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 ; Ld2 and each Ld1 are independently a chemical bond; or selected from -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, said natural amino acid or oligomeric natural amino acid being independently unsubstituted or substituted on the side chain with -(PEG) j -R 11 replace; -(PEG) t -、-(PEG) i -and-(PEG) j - each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(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 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 it is attached, it forms a 3-6 membered cycloalkyl group; or R 3 and R 4 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group; R 11 It is C 1-10 alkyl; m is any integer from 1 to 3; n is any integer from 2 to 20; d is 0, or any integer from 1 to 6; Each i is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i is independently an integer from 0 to 12; more preferably an integer from 0 to 8; in particular 4; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably an integer from 8 to 12; in particular 8 or 12; Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably an integer from 8 to 12; in particular 8 or 12; A is the antibody or binding fragment thereof according to claim 1, which is preferably modified to be the same as G in formula (III). n Partially connected, and G is glycine; z is an integer of 1-20.

8. The conjugate according to claim 7, wherein The conjugate has the following structure: Preferably, z is 1 to 4; preferably 2; Each i, i1, i2, i3, i4 is independently an integer from 0 to 100, preferably an integer from 0 to 20; preferably, each i, i1, i2, i3, i4 is independently an integer from 0 to 12; more preferably an integer from 0 to 8; in particular 4; Each j is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably an integer from 8 to 12; in particular 8 or 12; Preferably, n is 3, L 2 Yes - (CH2) p -(CH2)2(CO)- or -(C2H4-O) p -(CH2)2(CO)-, p is 2 to 4, L 1 is Gly-Gly-Phe-Gly (SEQ ID NO: 45), B is -NH-CH2-U-, or is absent, or is -NH-CH2-U-(CR 1 R 2 ) g -(CO)-, U does not exist, or U is O, g is 1; Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably an integer from 8 to 12; in particular 8 or 12; m is any integer from 1 to 3; in particular 1 or 2.

9. The conjugate according to claim 7 or 8, wherein The cargo is a cytotoxin or a fragment thereof, which is optionally derivatized so as to be attached to the B moiety or L in the compound of formula (III) as defined in claim 7. 1 part; Preferably, the cytotoxin is selected from the group consisting of: taxanes; maytansines; auristatins; epothilones; phosphate compretin A-4, compretin A-4 and derivatives thereof; indole-sulfonamides; vinca alkaloids, such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vindesine, anhydrous vinblastine; dolastatin 10 and analogs; halichondrin B; eribulin; indole-3-oxoacetamide; podophyllotoxins; 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC); spondylolipids; lelicycin; camptothecins and derivatives thereof; mitoxantrone; mitoguanidine; nitrogen mustards; nitrosoureas; aziridines; benzodopa; carboquinone; metodepa; uredepa; danemycin; Esperamycin; Neocarcin; Aclarubicin; Actinomycin; Anthramycin; Bleomycin; Actinomycin C; Carrubicin; Carminomycin; Anticancer Mycin; Carminomycin; Actinomycin D; Daunorubicin; Detorubicin; Adriamycin; Epirubicin; Esorubicin; Idarubicin; Mexiromycin; Mitomycin; Noramycin;Olivomycin; Peplomycin; Porfiromycin; Puromycin; Ferroadriamycin; Rhodorubicin; Streptomelanocin; streptozotocin; linastatin; levorubicin; trichothecenes; T-2 toxin; mucin A; baculosporin A; serpentine; ubenimex; azaserine; 6-diazo-5-oxo-L-norleucine; dimethylfolate; methotrexate; pteropterin; trimetrexate; edatrexate; fludarabine; 6-mercaptopurine; thiopurine; thioguanine; ancitabine; gemcitabine; enocitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; floxuridine; dimethyltestosterone; drostanolone propionate; cyclothiosteroid; melastane; testolactone; aminoglutethimide; mitotane; trilostane; flutamide; nilutamide; bicalutamide; leuprolide acetate; protein kinase inhibitors; and proteasome inhibitors; and / or Selected from vinca alkaloids, colchicines, taxanes, auristatins, maytansines, calicheamicin, doxorubicin, duocarmycin, SN-38, Nostoc cyclopeptide analogs, derutecan, duocarbamazine, calicheamicin, cintamycin, dolatamcin, pyrrolobenzodiazepine, exatecan and derivatives thereof; and / or Selected from auristatins, in particular MMAE, MMAF or MMAD; and / or Selected from exatecan and its derivatives, such as DX8951f.

10. The conjugate according to any one of claims 7 to 9, wherein The supported substance has a structure of formula (i): in a* is 0 or 1; The carbon atoms labeled p1* and p2* are each an asymmetric center, and the asymmetric center is in the S configuration, the R configuration, or racemic; L 1* Selected from C 1-6 Alkylene, which is unsubstituted or substituted with one substituent selected from halogen, -OH and -NH2; M* is -CH2-, -NH- or -O-; L 2* It is C 1-3 Alkylene; R 1* and R 2* are each independently selected from hydrogen, C 1-6 Alkyl, halogen and C 1-6 Alkoxy; Preferably, The load is selected from Especially selected from 11. The conjugate according to any one of claims 7 to 9, wherein the conjugate is selected from Each g is independently an integer from 1 to 6, preferably an integer from 1 to 3; more preferably 1; Each R 1 and R 2 are 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 it is attached, it forms a 3-6 membered cycloalkyl group; preferably, R 1 and R 2 It is hydrogen; Each t is independently an integer from 1 to 100, preferably an integer from 1 to 20; preferably, each t is independently an integer from 1 to 12; more preferably an integer from 8 to 12; in particular 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. A pharmaceutical composition comprising a preventive or therapeutically effective amount of the antibody or antigen-binding fragment according to any one of claims 1 to 4, the conjugate according to any one of claims 7 to 11, the polynucleotide or vector according to claim 5, or the host cell according to claim 6; and at least one pharmaceutically acceptable carrier.

13. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 4, the conjugate according to any one of claims 7 to 11, or the pharmaceutical composition according to claim 12 in the manufacture of a medicament for treating a disease; wherein the disease is a TROP2-related tumor, and the TROP2-related tumor includes a tumor that overexpresses TROP2 or a tumor with a TROP2 gene mutation, preferably, the tumor includes breast cancer, gastric cancer, lung cancer, ovarian cancer and urothelial cancer.

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