Antibody-drug conjugate comprising antibody binding to antibody against human cldn18.2 and its use

Abstract

This invention relates to novel antibody-drug conjugates (ADCs) targeting claudin18 isoform 2 (CLDN18.2), active metabolites of such ADCs, methods for preparing such ADCs, and the use of such ADCs for the treatment and / or prevention of diseases. More specifically, it relates to the use of such ADCs in the production of medicaments for the treatment and / or prevention of diseases, more specifically, diseases of excessive proliferation and / or angiogenesis such as cancer. More particularly, it relates to ADCs comprising an antibody or an antigen-binding fragment thereof that binds to CLDN18.2, and pharmaceutical compositions comprising the same.

Description

Technical Field

[0001] The present invention relates to a novel antibody-drug conjugate (ADC) targeting claudin 18 isoform 2 (CLDN18.2), an active metabolite of such an ADC, a method for preparing such an ADC, and the use of such an ADC for treating and / or preventing diseases. More specifically, it relates to the use of such an ADC in the production of a medicament for treating and / or preventing diseases, more specifically, hyperproliferative and / or angiogenic diseases such as cancer. More specifically, it relates to an ADC comprising a novel antibody or antigen-binding fragment thereof that binds to CLDN18.2, and a pharmaceutical composition comprising the same. Prior Art

[0002] Cancer is a disease caused by the uncontrolled growth of tissues, resulting in abnormal masses. It is the result of unregulated cell growth in various tissues. Early-stage tumors can be removed through surgery and radiation therapy, while metastatic tumors are usually treated with chemotherapy for palliative care.

[0003] As a result of systemic administration, most chemotherapeutic agents administered parenterally can induce adverse side effects and even have severe effects that induce toxicity. Therefore, efforts are being made to develop new chemotherapeutic agents that can improve and selectively apply such chemotherapeutic agents to tumor cells or adjacent tissues while achieving enhanced efficacy and minimizing toxicity / side effects.

[0004] Antibody-drug conjugates (ADCs) are a new targeted technology that binds toxins or drugs to antigen-binding antibodies, releasing toxic substances inside cells to induce apoptosis of cancer cells. Because they precisely deliver drugs to targeted cancer cells while minimizing effects on healthy cells and release them only under specific conditions, they offer superior efficacy compared to antibody therapeutics alone and significantly reduce the risk of side effects compared to existing anticancer agents.

[0005] The basic structure of this antibody-drug conjugate consists of "antibody-linker-small molecule drug (toxin)." The linker not only functions to connect the antibody and the drug, but also, after circulating in the body and stably reaching the target cell, the antibody-drug conjugate enters the cell. Due to the dissociation phenomenon between the antibody and the drug (e.g., enzymatic degradation), the drug is easily released and expresses its efficacy against the targeted cancer cells. In other words, the stability of the linker plays a very important role in the safety aspects of the antibody-drug conjugate, such as efficacy and systemic toxicity (Discovery Medicine 2010, 10(53):329-39). 85-82018-08-14).

[0006] The patent filed by the inventors of this invention (Korean Patent No. 1628872) discloses a linker containing a self-immolative group. This group is not only more stable in plasma but also stable during in vivo circulation. Furthermore, the drug is readily released within cancer cells, allowing for its efficacy. Meanwhile, considerable success has been achieved in the use of monoclonal antibodies for cancer treatment. Monoclonal antibodies are suitable for targeting tumor tissues and cells. Antibody-drug conjugates have become a powerful new treatment option for lymphomas and solid cancers, and immunomodulatory antibodies have recently achieved considerable clinical success. The development of therapeutic antibodies is based on a deep understanding of cancer serology, protein engineering technologies and their effects, mechanisms of drug resistance, and the interaction between the immune system and cancer cells.

[0007] Antigens expressed on the surface of human cancer cells are defined as a broad range of targets that are overexpressed, mutated, or selectively expressed compared to normal tissues. A key challenge lies in identifying antigens suitable for antibody-based therapeutics. These therapeutics mediate alterations in antigen or receptor function (i.e., acting as stimulators or antagonists) and modulate the immune system through Fc and T cell activation. Their efficacy is achieved through the delivery of specific drugs bound to antibodies targeting specific antigens. Molecular technologies that can modify antibody pharmacokinetics, efficacy, size, and immunostimulatory properties are becoming key elements in the development of new antibody-based therapies. Evidence from clinical trials of therapeutic antibodies in cancer patients emphasizes the importance of optimizing antibody selection strategies, including affinity and binding between the target antigen and the antibody, choice of antibody structure, and therapeutic approach (blocking signaling or immune efficacy).

[0008] Claudin 18 isoform 2 (CLDN18.2), a transmembrane membrane protein, consists of 261 amino acids in full length, two extracellular loops, and four transmembrane domains. Compared with CLDN18.1 expressed in some normal tissues, the partial amino acid sequences are different up to extracellular domain loop 1.

[0009] CLDN18.2 belongs to the claudin family, which is an important structural element of tight junctions. Claudin proteins are expressed in various cancer tissues and are potential targets for diagnostic and treatment methods. Moreover, it is known to play an important role in tumor formation and inflammatory responses. Changes in claudin expression not only induce dysfunction of tight junctions and affect signal transduction pathways, but also promote tumor growth in some epithelial cancers. It has been reported that in addition to being expressed in the apical region of some gastric mucosa, CLDN18.2 is also specifically expressed in primary and metastatic gastric cancers. As a cancer-specific antigen related to malignant transformation, it is also ectopically activated in pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, etc.

[0010] Under the above-mentioned technical background, the inventors of the present invention dedicated themselves to developing an antibody that is different from CLDN18.1 expressed in normal tissues and specifically binds to CLDN18.2 that expresses tumor specificity. As a result, an anti-CLDN18.2 antibody showing excellent binding affinity to CLDN18.2 was developed. To further enhance the effect of the antibody, a linker containing a self-immolative group was applied to the anti-cancer therapeutic antibody that specifically binds to CLDN18.2. Since the above-mentioned self-immolative group is more stable in plasma and also stable during circulation in the body, the drug is easily released in cancer cells and can effectively express its medicinal effect. Thus, a novel antibody-drug conjugate (ADC) targeting CLDN18.2 that can effectively treat and / or prevent hyperproliferative and / or metastatic cancer diseases can be provided, thereby completing the present invention. Summary of the Invention Problems to be Solved by the Invention

[0011] An object of the present invention is to provide a novel antibody-linker drug conjugate targeting claudin 18 isoform 2 (CLDN18.2) or a salt or solvate thereof.

[0012] Another object of the present invention is to provide an antibody-drug conjugate comprising an antibody that specifically binds to CLDN18.2 and a drug bound thereto, and a pharmaceutical composition comprising the same.

[0013] Another object of the present invention is to provide an antibody-linker-drug (toxin) system as described below, which combines a linker technology that contains a self-shedding group that is more stable in plasma and even stable when circulating in the body, and facilitates the release of the drug in cancer cells to maximize its efficacy, thereby allowing the drug and / or toxin to stably reach the target cells and effectively exert its efficacy while significantly reducing toxicity. Problem solving method

[0014] In one embodiment of the present invention, the provided conjugate or its pharmaceutically acceptable salt or solvate is represented by the following general formula I:

[0015] Formula I

[0016] Ab-[LINKER-(B)l]m

[0017] In the above general formula,

[0018] Ab is an antibody or antigen-binding fragment that specifically binds to claudin 18 isoform 2 (CLDN18.2) comprising the heavy chain variable region and the light chain variable region.

[0019] The heavy chain variable region comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0020] The light chain variable region comprises a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 13.

[0021] LINKER is a linker,

[0022] B is the active agent part,

[0023] l and m are each independently an integer selected from 1 to 20,

[0024] When l is an integer greater than or equal to 2, B may be the same or different.

[0025] In one embodiment of the present invention, the antibody or antigen-binding fragment that specifically binds to CLDN18.2 (claudin 18 isoform 2) comprises a heavy chain variable region and a light chain variable region.

[0026] The heavy chain variable region comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0027] The light chain variable region includes a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 13.

[0028] Furthermore, the heavy chain variable region comprises a human antibody heavy chain FR, and the gene encoding the heavy chain FR may be derived from the germline V gene IGHV3-23. Specifically, the heavy chain FR may comprise a heavy chain FR1 composed of the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain FR2 composed of the amino acid sequence set forth in SEQ ID NO: 3, a heavy chain FR3 composed of the amino acid sequence set forth in SEQ ID NO: 5, and a heavy chain FR4 composed of the amino acid sequence set forth in SEQ ID NO: 7.

[0029] Furthermore, the light chain variable region comprises a light chain FR of a human antibody, and the gene encoding the light chain FR may be derived from the germline V gene IGKV3-11 or the germline V gene IGKV3-15. Specifically, the light chain FR may comprise a light chain FR1 composed of the amino acid sequence set forth in SEQ ID NO: 8, a light chain FR2 composed of the amino acid sequence set forth in SEQ ID NO: 10, a light chain FR3 composed of the amino acid sequence set forth in SEQ ID NO: 12, and a light chain FR4 composed of the amino acid sequence set forth in SEQ ID NO: 14.

[0030] Furthermore, in order to bind to the linker, the light chain may include a portion consisting of the amino acid sequence represented by SEQ ID NO: 19 at the C-terminus.

[0031] In one embodiment of the present invention, the antibody or antigen-binding fragment that specifically binds to the above-mentioned CLDN18.2 comprises: a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence having more than 80% homology thereto; and a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 16 or an amino acid sequence having more than 80% homology thereto.

[0032] More specifically, the antibody or antigen-binding fragment that specifically binds to the above-mentioned CLDN18.2 comprises: a heavy chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 15, or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 95% or more homology thereto; and a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO: 16, or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 95% or more homology thereto, which specifically binds to CLDN18.2.

[0033] More specifically, the antibody or antigen-binding fragment that specifically binds to CLDN18.2 comprises: the amino acid sequence represented by SEQ ID NO: 15, or an antibody or antigen-binding fragment thereof having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 100% or more of the amino acid sequence represented by SEQ ID NO: 15. and a light chain variable region consisting of an amino acid sequence represented by SEQ ID NO:16 or an amino acid sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity thereto, which specifically binds to CLDN18.2.

[0034] In one embodiment of the present invention, the antibody or antigen-binding fragment that specifically binds to the above-mentioned CLDN18.2 comprises: a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 17 or an amino acid sequence having more than 80% homology thereto; and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 18 or an amino acid sequence having more than 80% homology thereto.

[0035] More specifically, the antibody or antigen-binding fragment that specifically binds to CLDN18.2 comprises: a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 95% or more homology thereto; and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence having 80% or more, 85% or more, 90% or more, or 95% or more homology thereto, which specifically binds to CLDN18.2.

[0036] More specifically, the antibody or antigen-binding fragment that specifically binds to CLDN18.2 comprises: the amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity thereto. and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18, or an amino acid sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more identity thereto, which specifically binds to CLDN18.2.

[0037] In one example, the heavy chain consisting of an amino acid sequence having at least 80% homology to the amino acid sequence represented by SEQ ID NO: 17 comprises L237A and L238A mutations.

[0038] In one embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment thereof can be one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single-chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single-chain variable antibody (scFv), a scFv-Fc fragment, a single-domain heavy chain antibody, a single-domain light chain antibody, an antibody variant, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

[0039] In this specification, "linker" refers to a compound that covalently bonds a cytotoxic compound to a ligand.

[0040] In the present invention, the described linker can be cleavable, non-cleavable, hydrophilic or hydrophobic.

[0041] Under intracellular conditions, the cleavable linker can be cleaved, thereby releasing the active agent from the antibody construct-active agent conjugate in the intracellular environment through cleavage of the linker.

[0042] Cleavable linkers can be cleaved by cleavage agents present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). For example, cleavable linkers include lysosomal proteases or endosomal proteases, but are not limited thereto and can also be peptidyl linkers cleaved by intracellular peptidases or proteases. Typically, such peptidyl linkers are at least two amino acids in length or at least three amino acids in length. Cleavage agents can include cathepsin B, cathepsin D, and plasmin, all of which are known to release active drugs within target cells by hydrolyzing dipeptide drug derivatives (e.g., see Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most common peptidyl linkers are those cleaved by enzymes present in antigen-expressing cells. For example, peptidyl linkers cleaved by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues (e.g., Phe-Leu linkers or Gly-Phe-Leu-Gly linkers) can be used. For example, other linkers are described in U.S. Patent No. 6,214,345. Furthermore, the peptidyl linker cleavable by intracellular proteases may be a Val-Cit linker, a Phe-Lys linker (for example, see the doxorubicin synthesized using the Val-Cit linker described in U.S. Patent No. 6,214,345), or a Val-Ala linker. The Val-Cit linker or Val-Ala linker may contain a pentafluorophenyl group, a succinimidyl group, or a maleimido group. Alternatively, it may contain a 4-aminobenzoic acid group (PABA) and a pentafluorophenyl group, a 4-aminobenzoic acid group (PABA) and a maleimido group, or a 4-aminobenzoic acid group and a succinimidyl group.

[0043] As used herein, the terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction within a cell that results in the covalent attachment of the active agent B to the antibody construct Ab, e.g., the generation of other metabolites of the conjugate that are separated from the free drug or antibody within the cell due to the destruction of the linker.

[0044] Furthermore, the cleavable linker is pH-sensitive, that is, it can be easily hydrolyzed at a specific pH value. Generally, the pH-sensitive linker can be hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) can be hydrolyzed in lysosomes (e.g., see U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). While this linker is relatively stable at neutral pH, such as in blood, it is unstable at lysosomal pH levels below pH 5.5 or 5.0. An example of a hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond (e.g., see U.S. Patent No. 5,622,929)).

[0045] Furthermore, the linker can be cleaved under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers are known, including disulfide linkers that can be formed using SATA (N-succinimidyl-5-acetylmuroacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)propionate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-thio)toluene), SPDB, and SMPT (e.g., see U.S. Patent No. 4,880,935, Thorpe et al., 1987, Cancer Res. 47:5924-5931).

[0046] Furthermore, the linker can be a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12), a β-glucuronide linker (Jeffery et al., 2006, Bioconjug Chem. 17(3): 832-40), or a β-galactosidase linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15; 142: 376-382).

[0047] The non-cleavable linker may be a maleimidohexanoyl linker. The maleimidohexanoyl linker may comprise N-maleimidomethylcyclohexane-1-carboxylate. The maleimidohexanoyl linker may comprise a succinimidyl group. The maleimidohexanoyl linker may comprise a pentafluorophenyl group. The linker may be a combination of a maleimidohexanoyl group and one or more polyethylene glycol molecules. The linker may be a combination of a maleimidohexanoyl group and one or more polyethylene glycol molecules. The linker may be a maleimido-PEG4 linker. The linker may be a combination of a maleimidohexanoyl linker containing a succinimidyl group and one or more polyethylene glycol molecules. The linker may be a combination of a pentafluorophenyl group and a maleimidohexanoyl linker containing one or more polyethylene glycol molecules. The linker may comprise a maleimide linked to a polyethylene glycol molecule, wherein the polyethylene glycol may allow for greater linker flexibility or the use of longer linkers. The linker may be a (maleimidohexanoyl)-(valine-citrulline)-(p-aminobenzyloxycarbonyl) linker.

[0048] In one embodiment of the present invention, the linker can be a cleavable linker.

[0049] In one embodiment of the present invention, the linker can be a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, a self-immolative linker, a self-stabilizing linker, a malonate linker, a maleimidobenzyl linker, a 3'-N-amide analog, a β-glucuronide linker, or a β-galactoside linker.

[0050] In one embodiment of the present invention, the protease-cleavable linker may comprise a thiol-reactive spacer or a dipeptide. More specifically, the protease-cleavable linker may comprise a thiol-reactive maleimidohexanoyl spacer, a valine-citrulline dipeptide, or a p-amino-benzyloxycarbonyl spacer.

[0051] In one embodiment of the present invention, the acid-cleavable linker may be a hydrazine linker or a quaternary ammonium linker.

[0052] In one embodiment of the present invention, the linker may have a structure of Formula II.

[0053] Formula II

[0054]

[0055] In the above general formula,

[0056] The wavy line indicates the site where the antibody or antigen-binding fragment binds specifically to CLDN18.2, and the * indicates the site where the active agent binds.

[0057] G is a glucuronic acid moiety or , the above R3 is hydrogen or a carboxyl protecting group, the above R4 are each independently hydrogen or a hydroxyl protecting group,

[0058] R1 and R2 are each independently hydrogen, C1-8 alkyl or C3-8 cycloalkyl,

[0059] W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'- or -PO2NR'-, the above C, S or P is directly bonded to a phenyl ring, and the above R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamino or di-C1-8 alkylamino, C3-20 heteroaryl or C6-20 aryl.

[0060] Z is independently hydrogen, C1-8 alkyl, halogen, cyano or nitro,

[0061] n is an integer from 0 to 3,

[0062] L is one of the following A) or B),

[0063] A) C1-50 alkylene or 1-50 membered heteroalkylene, satisfying one or more of the following conditions:

[0064] (i) L contains one or more unsaturated bonds;

[0065] (ii) 2 atoms in L are substituted with a divalent substituent such as a substituent, which completes the heteroarylene;

[0066] (iii) L is a 1- to 50-membered heteroalkylene group;

[0067] (iv) the above alkylene group is substituted by one or more C1-20 alkyl groups;

[0068] B) contains one or more isoprene derivative units that can be recognized by isoprenoid transferase, represented by the following general formula III:

[0069] Formula III

[0070]

[0071] In one embodiment of the present invention, the conjugate may have the following structure of Formula IIa:

[0072] Formula IIa

[0073]

[0074] In the above general formula,

[0075] Ab is an antibody or antigen-binding fragment that specifically binds to CLDN18.2 comprising the heavy chain variable region and the light chain variable region,

[0076] B' are each independently the same or different active agents,

[0077] G is each independently a glucuronic acid moiety or

[0078] The above R3 is hydrogen or a carboxyl protecting group, and the above R4 are each independently hydrogen or a hydroxyl protecting group,

[0079] R1 and R2 are each independently hydrogen, C1-8 alkyl or C3-8 cycloalkyl,

[0080] W is each independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'- or -PO2NR'-, the above C, S or P is directly bonded to a phenyl ring, the above NR' is bonded to L, and the above R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamino or di-C1-8 alkylamino, C3-20 heteroaryl or C6-20 aryl,

[0081] Z is each independently hydrogen, C1-8 alkyl, halogen, cyano or nitro,

[0082] n is an integer from 0 to 3. When n is an integer of 2 or greater, each Z may be the same or different.

[0083] L is each independently one of the following A) or B),

[0084] A) C1-50 alkylene or 1-50 membered heteroalkylene, satisfying one or more of the following conditions:

[0085] (i) L contains one or more unsaturated bonds;

[0086] (ii) 2 atoms in L are substituted with a divalent substituent such as a substituent, which completes the heteroarylene;

[0087] (iii) L is a 1- to 50-membered heteroalkylene group;

[0088] (iv) the above alkylene group is further substituted by one or more C1-20 alkyl groups;

[0089] B) contains one or more isoprene derivative units that can be recognized by isoprenoid transferase, represented by the following general formula III:

[0090] Formula III

[0091]

[0092] m is an integer selected from 1 to 20.

[0093] Furthermore, the present invention relates to a conjugate represented by the following general formula IIa or a pharmaceutically acceptable salt or solvate thereof.

[0094] Formula IIa

[0095]

[0096] In the above general formula,

[0097] Ab is an antibody or antigen-binding fragment that specifically binds to claudin 18 isoform 2 (CLDN18.2).

[0098] G is each independently a glucuronic acid moiety or

[0099] The above R3 is hydrogen or a carboxyl protecting group, and the above R4 are each independently hydrogen or a hydroxyl protecting group,

[0100] R1 and R2 are each independently hydrogen, C1-8 alkyl or C3-8 cycloalkyl,

[0101] W is each independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R"NR'-, -SONR'- or -PO2NR'-, the above C, S or P is directly bonded to a phenyl ring, the above NR' is bonded to L, and the above R' and R" are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamino or di-C1-8 alkylamino, C3-20 heteroaryl or C6-20 aryl,

[0102] Z is each independently hydrogen, C1-8 alkyl, halogen, cyano or nitro,

[0103] n is an integer from 0 to 3,

[0104] L is each independently one of the following A) or B),

[0105] A) C1-50 alkylene or 1-50 membered heteroalkylene, satisfying one or more of the following conditions:

[0106] (i) L contains one or more unsaturated bonds;

[0107] (ii) 2 atoms in L are substituted with a divalent substituent such as a substituent, which completes the heteroarylene;

[0108] (iii) L is a 1- to 50-membered heteroalkylene group;

[0109] (iv) the above alkylene group is substituted by one or more C1-20 alkyl groups;

[0110] B) contains one or more isoprene derivative units that can be recognized by isoprenoid transferase, represented by the following general formula III:

[0111] Formula III

[0112]

[0113] B' is the active agent,

[0114] Ab is bound to the part indicated by the wavy line,

[0115] l and m are each independently an integer selected from 1 to 20,

[0116] When l is an integer greater than 2, the active agents can be the same or different.

[0117] In one embodiment of the present invention, the above-mentioned G is a glucuronic acid moiety or a compound of the following chemical formula IIIa:

[0118] Chemical formula IIIa

[0119]

[0120] in,

[0121] R3 is hydrogen or a carboxyl protecting group,

[0122] Each R4 is independently hydrogen or a hydroxy protecting group.

[0123] In one embodiment of the present invention, the above G are each independently

[0124] R3 is hydrogen or a carboxyl protecting group,

[0125] The above R4 are each independently hydrogen or a hydroxyl protecting group.

[0126] In one embodiment of the present invention, the above R3 is hydrogen, and each R4 is hydrogen.

[0127] In one embodiment of the present invention, R1 and R2 are each hydrogen.

[0128] Furthermore, each of the above Zs is independently hydrogen, C1-8 alkyl, halogen, cyano or nitro.

[0129] Furthermore, the above-mentioned W is -C(O)-, -C(O)NR'-, or -C(O)O-. More specifically, the above-mentioned W is -C(O)NR'-, wherein C(O) is connected to the benzene ring and NR' is bonded to L. The above-mentioned R' is hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamino or di-C1-8 alkylamino, C3-20 heteroaryl, or C6-20 aryl.

[0130] Furthermore, the above n is 0, 1, 2 or 3, more specifically 0, 1 or 2, and further specifically 0.

[0131] More specifically, the above G is a compound of the following chemical formula IIIa:

[0132] Chemical formula IIIa

[0133]

[0134] in,

[0135] R3 is hydrogen or a carboxyl protecting group,

[0136] Each R4 is independently hydrogen or a hydroxyl protecting group,

[0137] W is -C(O)NR'-, wherein C(O) is connected to the benzene ring, NR' is bonded to L, each Z is a C1-8 alkyl, halogen, cyano or nitro, n is 0, m is 1, and R1 and R2 are hydrogen.

[0138] In one embodiment of the present invention, at least one L is an alkylene group having 1 to 100 carbon atoms, wherein the carbon atoms of the alkylene group may be substituted by one or more heteroatoms selected from the group consisting of N, O and S, and the alkylene group may be further substituted by one or more alkyl groups having 1 to 20 carbon atoms.

[0139] Specifically, at least one L is a C1-50 alkylene or a 1-50 membered heteroalkylene, and may satisfy one or more of the following conditions:

[0140] (i) contains one or more unsaturated bonds;

[0141] (ii) 2 atoms in L are substituted with a divalent substituent such as a substituent, which completes the heteroarylene;

[0142] (iii) L is a 1- to 50-membered heteroalkylene group;

[0143] (iv) The above alkylene group is substituted by one or more C1-20 alkyl groups.

[0144] Furthermore, at least one of the above Ls is a nitrogen-containing 1-50 membered heteroalkylene group, the linker comprises two or more atoms of a hydrophilic amino acid, and the above nitrogen can form a peptide bond with the carbonyl group of the hydrophilic amino acid.

[0145] In one embodiment of the present invention, L is C2-50 alkylene, C2-50 heteroalkylene, hydrophilic amino acid, -C(O)-, -C(O)NR"-, -C(O)O-, -(CH2)s-NHC(O)-(CH2)t-, -(CH2)uC(O)NH-(CH2)v-, -(CH2)s-NHC(O)-(CH2)tC(O)-, -(CH2)uC(O)NH-(CH2)vC(O)-, -S(O)2NR"-, -P(O)R'''NR"-, -S(O)NR"- or -PO2NR"-,

[0146] wherein R″ and R''' are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamino or bis-C1-8 alkylamino, C3-20 heteroaryl or C5-20 aryl, and s, t, u and v are each independently an integer from 0 to 10.

[0147] In one embodiment of the present invention, the above-mentioned L is a nitrogen-containing 1-5 membered heteroalkylene group, the above-mentioned linker contains two or more atoms of a hydrophilic amino acid, and the above-mentioned nitrogen forms a peptide bond with the carbonyl group of the hydrophilic amino acid.

[0148] In one embodiment of the present invention, the L is covalently bonded to the antibody via a thioether bond, wherein the thioether bond comprises a sulfur atom of a cysteine residue of the antibody.

[0149] In one embodiment of the present invention, at least one L is a hydrophilic amino acid.

[0150] In one embodiment of the present invention, the hydrophilic amino acid may be arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine or threonine.

[0151] In one embodiment of the present invention, the hydrophilic amino acid may be an amino acid having a side chain having a residue that is charged under neutral pH conditions in an aqueous solution.

[0152] In one embodiment of the present invention, the hydrophilic amino acid is aspartic acid or glutamic acid.

[0153] In one embodiment of the present invention, the hydrophilic amino acid is ornithine or lysine.

[0154] In one embodiment of the present invention, the hydrophilic amino acid is arginine.

[0155] In one embodiment of the present invention, at least one L is -C(O)-, -C(O)NR"-, -C(O)O-, -S(O)2NR"-, -P(O)R'''NR"-, -S(O)NR"- or -PO2NR"-, and R" and R''' are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamino or bis-C1-8 alkylamino, C3-20 heteroaryl or C6-20 aryl.

[0156] In one embodiment of the present invention, at least one L is -C(O)NR"-, -(CH2)s-NHC(O)-(CH2)t-, -(CH2)uC(O)NH-(CH2)v-, -(CH2)s-NHC(O)-(CH2)tC(O)- or -(CH2)uC(O)NH-(CH2)vC(O)-, wherein R" is hydrogen, C1-5 alkyl, C3-8 cycloalkyl, C1-5 alkoxy, C3-20 heteroaryl or C6-20 aryl, and s, t, u and v are each independently an integer from 0 to 10.

[0157] In one embodiment of the present invention, at least one L is -C(O)NR"-, and R" is hydrogen.

[0158] In one embodiment of the present invention, at least one L is -(CH2)s-NHCO-, where s is an integer from 1 to 5.

[0159] In one embodiment of the present invention, at least one L is -(CH2)uC(O)NH-(CH2)vC(O)-, where u is an integer from 1 to 5 and v is an integer from 1 to 5.

[0160] In one embodiment of the present invention, L comprises 、

[0161] Wherein, L1 is a direct bond or an alkylene group having 1 to 30 carbon atoms, R11 is hydrogen or an alkyl group having 1 to 10 carbon atoms, and L2 is an alkylene group having 1 to 30 carbon atoms.

[0162] In one embodiment of the present invention, the above L further comprises a second unit represented by Formula VIII or Formula IX.

[0163] In one embodiment of the present invention, at least one second unit is represented by Formula VIII or Formula IX:

[0164] Formula VIII

[0165] -(CH2)r(V(CH2)p)q-

[0166] Formula IX

[0167] -(CH2CH2X)w-

[0168] The above V is a single bond, -O-, -S-, -NR21-, -C(O)NR22-, -NR23C(O)-, -NR24SO2- or -SO2NR25-,

[0169] X is -O-, C1-8 alkylene or -NR21-,

[0170] R21 to R25 are each independently hydrogen, C1-6 alkyl, C1-6 alkyl C6-20 aryl or C1-6 alkyl C3-20 heteroaryl,

[0171] r is an integer from 0 to 10,

[0172] p is an integer from 0 to 10,

[0173] q is an integer from 1 to 20,

[0174] w is an integer from 1 to 20.

[0175] In one embodiment of the present invention, q may be 4 to 20, more specifically 5 to 20. Furthermore, q may be 1 to 10 or 2 to 12, more specifically 2, 4, 5, or 11. Furthermore, r may be 1 or 2. Furthermore, p may be 1 or 2. Furthermore, V may be -O-.

[0176] More specifically, r is 2, p is 2, q is 2, 4, 5 or 11, and V may be -O-.

[0177] Furthermore, in one embodiment of the present invention, X may be -O-. Furthermore, w may be an integer from 4 to 20.

[0178] More specifically, X is -O-, and w can be 1 to 10 or 4 to 20.

[0179] In one embodiment of the present invention, at least one second unit is at least one polyethylene glycol unit, the structure of which is or , where n is 1 to 12.

[0180] In one embodiment of the present invention, at least one second unit is 1 to 12-OCH2CH2-units, 3 to 12-OCH2CH2-, 5 to 12-OCH2CH2-units, 6 to 12-OCH2CH2-units or 3-OCH2CH2-units.

[0181] In one embodiment of the present invention, at least one second unit is -(CH2CH2X)w-,

[0182] Wherein, X is a single bond, -O-, C1-8 alkylene or -NR21-,

[0183] R21 is hydrogen, C1-6 alkyl, C1-6 alkyl-C6-20 aryl or C1-6 alkyl-C3-20 heteroaryl, and w is an integer from 1 to 20, specifically 1, 3, 6 or 12.

[0184] In one embodiment of the present invention, X is -O-, and w is an integer from 6 to 20.

[0185] Specifically, in one embodiment of the present invention, the above-mentioned L comprises oxime, and at least one polyethylene glycol unit enables the oxime to be covalently bonded to the active agent.

[0186] In one embodiment of the present invention, the linker comprises a third unit formed by 1,3-dipolar cycloaddition reactions, hetero-Diels-Alder reactions, nucleophilic substitution reactions, non-aldol type carbonyl reactions, addition to carbon-carbon multiple bonds, osidation reactions, or click reactions.

[0187] In one embodiment of the present invention, the third unit can be formed by the reaction between an alkyne and an azide or the reaction between an aldehyde or ketone group and a hydrazine or an alkoxyamine.

[0188] In one embodiment of the present invention, the above L further comprises a third unit represented by the following formula IVa, IVb, IVc, IVd or IVe:

[0189] General formula Iva

[0190]

[0191] Formula IVb

[0192]

[0193] Formula IVc

[0194]

[0195] Formula IVd

[0196]

[0197] Formula IVe

[0198]

[0199] In the above general formula, L1 is a single bond or an alkylene group having 1 to 30 carbon atoms,

[0200] R11 is hydrogen or C1-10 alkyl.

[0201] In one embodiment of the present invention, the third unit comprises

[0202] in,

[0203] L1 is a single bond or an alkylene group having 1 to 30 carbon atoms, preferably an alkylene group having 10, 11, 12, 13, 14, 15 or 16 carbon atoms,

[0204] R11 is hydrogen or an alkyl group having 1 to 10 carbon atoms, specifically a methyl group,

[0205] L2 is an alkylene group having 1 to 30 carbon atoms.

[0206] In one embodiment of the present invention, L1 and L2 are each independently a single bond or a C1-30 alkylene group, and R11 is hydrogen or a C1-10 alkyl group.

[0207] In one embodiment of the present invention, L1 is a single bond or an alkylene group having 11 carbon atoms or an alkylene group having 12 carbon atoms.

[0208] Furthermore, in one embodiment of the present invention, the third unit comprises

[0209] The above V is a single bond, -O-, -S-, -NR21-, -C(O)NR22-, -NR23C(O)-, -NR24SO2- or -SO2NR25-,

[0210] R21 to R25 are each independently hydrogen, C1-6 alkyl, C1-6 alkyl C6-20 aryl or C1-6 alkyl C3-20 heteroaryl,

[0211] r is an integer from 1 to 10,

[0212] p is an integer from 0 to 10,

[0213] q is an integer from 1 to 20,

[0214] L1 is a single bond.

[0215] In one embodiment of the present invention, r may be 2 or 3. Furthermore, p may be 1 or 2. Furthermore, q may be 1 to 6.

[0216] More specifically, in the third unit, r is 2 or 3, p is 1 or 2, and q can be 1 to 6.

[0217] Furthermore, in one embodiment of the present invention, the linker comprising the third unit may be 、 , where the wavy line indicates the site of connection to the antibody construct, * indicates the site of connection to the active agent, and n is an integer from 0 to 20.

[0218] In one embodiment of the present invention, the third unit comprises

[0219] Furthermore, in one embodiment of the present invention, the linker comprises in the precursor state Furthermore, in the above structure, the third unit can be formed by a reaction between alkyne and azide, specifically, the third unit can be formed by a click reaction.

[0220] In one embodiment of the present invention, the linker comprises in a precursor state , a third unit can be formed by the above structure, and the third unit includes

[0221] In one embodiment of the present invention, the above-mentioned L is a 3- to 50-membered heteroalkylene group containing an oxime,

[0222] The oxygen atom of the above oxime is located on the side of L connected to W, and the carbon atom of the oxime is located on the side of L connected to Ab, or

[0223] The carbon atom of the oxime is located on the side of L connected to W, and the oxygen atom of the oxime is located on the side of L connected to Ab.

[0224] In one embodiment of the present invention, the above-mentioned L comprises oxime, and one or more isoprene units allow the oxime to be covalently bonded to Ab.

[0225] In one embodiment of the present invention, the linker may further comprise at least one isoprene unit, the structure of which is , where n is at least 2.

[0226] In one embodiment of the invention, at least one isoprene unit is a substrate for an isoprenoid transferase or a product of an isoprenoid transferase.

[0227] In one embodiment of the invention, the isoprene unit of the linker is covalently bonded to the antibody via a thioether bond comprising the sulfur atom of a cysteine of the antibody construct.

[0228] Furthermore, the isoprene unit allows the oxime contained in the linker to be covalently bonded to the antibody construct.

[0229] In one embodiment of the present invention, the antibody construct comprises an amino acid motif recognized by an isoprenoid transferase, and the thioether bond comprises the sulfur atom of a cysteine of the amino acid motif.

[0230] In one embodiment of the present invention, the antibody construct comprises an amino acid motif recognized by an isoprenoid transferase, and the thioether bond comprises the sulfur atom of a cysteine of the amino acid motif.

[0231] In one embodiment of the present invention, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC and CYYX, wherein C represents cysteine, Y independently represents an aliphatic amino acid in each occurrence, and X independently represents glutamine, glutamic acid, serine, cysteine, methionine, alanine or leucine in each occurrence, and the thioether bond comprises the sulfur atom of the cysteine of the amino acid motif.

[0232] In one embodiment of the present invention, the amino acid motif is a CYYX sequence, where Y is independently alanine, isoleucine, leucine, methionine or valine in each case.

[0233] In one embodiment of the present invention, the amino acid motif is a CVIM sequence or a CVLL sequence.

[0234] In one embodiment of the present invention, at least one of the 1 to 20 amino acids preceding the amino acid motif can be independently selected from glycine, arginine, aspartic acid and serine.

[0235] In one embodiment of the present invention, at least one of the 1 to 20 amino acids preceding the above amino acid motif is glycine.

[0236] In one embodiment of the present invention, one or more of the 1 to 10 amino acids preceding the amino acid motif can be independently selected from glycine, arginine, aspartic acid, and serine. For example, in one embodiment of the present invention, at least one of the 7 amino acids preceding the amino acid motif is glycine. Alternatively, three or more of the 7 amino acids preceding the amino acid motif are independently selected from glycine, arginine, aspartic acid, and serine. Alternatively, the 1 to 10 amino acids preceding the amino acid motif are glycine. Specifically, at least one, two, three, four, five, six, seven, eight, nine, or ten amino acids preceding the amino acid motif are glycine.

[0237] In one embodiment of the present invention, the antibody may comprise the amino acid sequence GGGGGGGCVIM.

[0238] In one embodiment of the present invention, the above-mentioned L may contain the amino acid sequence GGGGGGGCVIM at the C-terminus.

[0239] In yet another embodiment of the present invention, the Linker may include:

[0240] (a) more than one L; (b) more than one second unit; (c) more than one third unit; and (d) more than one fourth unit.

[0241] Among them, the second unit connects L and the third unit, L and the fourth unit, or the third unit and the fourth unit,

[0242] Among them, more than one fourth unit can release more than one drug or toxin.

[0243] Among them, L connects the second unit and the fourth unit, the second unit and the third unit, or the second unit and the other second unit.

[0244] In one embodiment of the present invention, the structure of the fourth unit is represented by the following chemical formula IIb:

[0245] Chemical formula IIb

[0246]

[0247] In the above chemical formula,

[0248] G is sugar, sugar acid or sugar derivatives,

[0249] W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O)2NR'-, -P(O)R”NR'-, -S(O)NR'- or -PO2NR'-,

[0250] In the case where C(O), S or P is directly connected to the benzene ring, R' and R" are each independently hydrogen, (C1-8) alkyl, (C3-8) cycloalkyl, (C1-8) alkoxy, (C1-8) alkylthio, mono-(C1-8) alkylamino or bis-(C1-8) alkylamino, (C3-20) heteroaryl or (C6-20) aryl, W is connected to L or the second unit,

[0251] Each Z is independently hydrogen, (C1-8) alkyl, halogen, cyano or nitro,

[0252] n is an integer from 1 to 3,

[0253] m is 0 or 1,

[0254] R1 and R2 are each independently hydrogen, (C1-8)alkyl or (C3-8)cycloalkyl, or R1 and R2 together with the carbon atoms to which they are attached form a (C3-8)cycloalkyl ring.

[0255] In one embodiment of the present invention, the sugar or sugar acid is a monosaccharide.

[0256] In one embodiment of the present invention, G is a compound of the following chemical formula IIIa:

[0257] Compound IIIa

[0258]

[0259] in,

[0260] R3 is hydrogen or a carboxyl protecting group,

[0261] Each R4 is independently hydrogen or a hydroxy protecting group.

[0262] In one embodiment of the present invention, R3 is hydrogen and each R4 is hydrogen.

[0263] In one embodiment of the present invention, W is -C(O)NR'-, wherein C(O) is connected to the benzene ring, and NR' is connected to L or the second unit.

[0264] In one embodiment of the present invention, Z is hydrogen.

[0265] In one embodiment of the present invention, R1 and R2 are each hydrogen.

[0266] In one embodiment of the present invention, the second unit is represented by -(CH2)r(V(CH2)p)q-, -((CH2)pV)q-, -(CH2)r(V(CH2)p)qY-, -((CH2)pV)q(CH2)r-, -Y((CH2)pV)q- or -(CH2)r(V(CH2)p)qYCH2-,

[0267] in,

[0268] r is an integer from 0 to 10,

[0269] p is an integer from 1 to 10,

[0270] q is an integer from 1 to 20,

[0271] V and Y are each independently a single bond, -O-, -S-, -NR21-, -C(O)NR22-, -NR23C(O)-, -NR24SO2- or -SO2NR25-,

[0272] R21 to R25 are each independently hydrogen, (C1-6)alkyl, (C1-6)alkyl(C6-20)aryl or (C1-6)alkyl(C3-20)heteroaryl.

[0273] In one embodiment of the present invention, r is 2.

[0274] In one embodiment of the present invention, p is 2.

[0275] In one embodiment of the present invention, q is an integer from 6 to 20.

[0276] In one embodiment of the present invention, q is 2, 5 or 11.

[0277] In one embodiment of the present invention, V and Y are each independently -O-.

[0278] In one embodiment of the present invention, L or the third unit comprises , wherein L1 is a direct bond or an alkylene group having 1 to 30 carbon atoms, R11 is hydrogen or an alkyl group having 1 to 10 carbon atoms, specifically a methyl group, and L2 is an alkylene group having 1 to 30 carbon atoms.

[0279] In one embodiment of the present invention, L1 is an alkylene group having 12 carbon atoms.

[0280] In one embodiment of the present invention, R11 is methyl.

[0281] In one embodiment of the present invention, L2 is an alkylene group having 11 carbon atoms.

[0282] In one embodiment of the present invention, the binding unit is covalently bonded to the antibody via a thioether bond comprising a sulfur atom of a cysteine residue of the antibody.

[0283] In one embodiment of the present invention, the antibody comprises an amino acid motif recognized by an isoprenoid transferase, and the thioether bond comprises the sulfur atom of a cysteine of the amino acid motif.

[0284] In one embodiment of the present invention, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC and CYYX, wherein C represents cysteine, Y independently represents an aliphatic amino acid in each occurrence, and X independently represents glutamine, glutamic acid, serine, cysteine, methionine, alanine or leucine in each occurrence, and the thioether bond comprises the sulfur atom of the cysteine of the amino acid motif.

[0285] In one embodiment of the present invention, the amino acid motif is a CYYX sequence, where Y is independently alanine, isoleucine, leucine, methionine or valine in each case.

[0286] In one embodiment of the present invention, the amino acid motif is a CVIM sequence or a CVLL sequence.

[0287] In one embodiment of the present invention, at least one of the 1 to 20 amino acids preceding the amino acid motif is glycine.

[0288] In one embodiment of the present invention, one or more of the 1 to 20 amino acids preceding the amino acid motif may be independently selected from glycine, arginine, aspartic acid and serine.

[0289] In one embodiment of the invention, 1 to 20 amino acids preceding the amino acid motif are glycine, specifically, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids preceding the amino acid motif are glycine.

[0290] In one embodiment of the present invention, the antibody may comprise the amino acid sequence GGGGGGGCVIM.

[0291] In one embodiment of the present invention, the isoprenoid transferase is farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase).

[0292] In one embodiment of the present invention, L comprises one or more branched linkers covalently bonded to Ab,

[0293] i) each branched linker comprises a fifth unit covalently bonded to Ab via a first linker PL,

[0294] ii) Each branched linker comprises a first branch B1, wherein the first active agent in the first branch is covalently bonded to the fifth unit via a second linker SL and a cleavage group CG,

[0295] iii) each branched linker further comprises: a) a second branch B2 in which the second active agent is covalently bonded to the fifth unit via a second linker SL and a cleavage group CG; or b) a second branch B2 in which a polyethylene glycol moiety is covalently bonded to the fifth unit,

[0296] Each of the above-mentioned cleavage groups can be hydrolyzed in order to release the active agent from the antibody construct-active agent conjugate.

[0297] In one embodiment of the present invention, i) the branched linker comprises a fifth unit BR covalently bonded to the reactive portion via the first linker PL,

[0298] ii) the fifth unit is covalently bonded to the first branch B1, and comprises a first active agent covalently bonded to the second linker SL and the cleavage group CG,

[0299] iii) a fifth unit covalently bonded to the second branch B2, comprising: a) a second active agent covalently bonded to a second linker SL and a cleavage group CG; or b) a polyethylene glycol portion, wherein each cleavage group can release the active agent by hydrolysis.

[0300] In one embodiment of the present invention, the cleavage group is as shown in the above chemical formula II.

[0301] In the present invention, the second linker (e.g., linking the active agent to the fifth unit) may include a third unit formed by a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, a carbon-carbon multiple bond addition reaction, an oxidation reaction, or a click reaction. The third unit may be formed by the reaction of an alkyne with an azide or a non-aldol carbonyl reaction as described above, for example, by the reaction of an aldehyde or ketone group with hydrazine or hydroxylamine, thereby achieving mild coupling of the active agent and / or cleavable group to L.

[0302] In one embodiment of the present invention, the fifth unit is 、

[0303] The above L2, L3, L4 are each independently a direct bond or -CnH2n-, and the above n is an integer from 1 to 30,

[0304] The above G1, G2, G4 are independently direct keys, 、

[0305] The above R30 is hydrogen or C1-30 alkyl,

[0306] The above R40 is hydrogen, C1-10 alkyl or -L5-COOR50, wherein L5 is a direct bond or -CnH2n-,

[0307] Wherein, n is an integer from 1 to 10, and R50 is hydrogen or a C1-30 alkyl group.

[0308] In one embodiment of the present invention, the fifth unit is 、

[0309] The above-mentioned L2, L3, L4, G1, G2, G3, R30, R40 and L5 may be the same as above.

[0310] In one embodiment of the present invention, the cleavage group can be cleaved within the target cell.

[0311] In one embodiment of the present invention, the cleavage group can release more than one active agent.

[0312] Furthermore, in one embodiment of the present invention, the conjugate comprises Ab,

[0313] Contains one, two, three or four or more branched linkers covalently bonded to Ab,

[0314] Each branched linker is combined with one or more active agents.

[0315] The active agents are the same or different.

[0316] Furthermore, in one embodiment of the present invention, the above-mentioned active agents are bonded to the branched linkers through cleavable bonds.

[0317] In one embodiment of the present invention, the aforementioned L is a linker comprising at least one fifth unit and a first linker PL.

[0318] In one embodiment of the present invention, the first linker of the antibody drug conjugate comprises an alkylene group having 1 to 100, preferably 1 to 50, carbon atoms, or

[0319] The alkylene group contains at least one unsaturated bond,

[0320] The alkylene group contains at least one heteroarylene group,

[0321] The carbon atoms of the alkylene group are substituted by one or more heteroatoms selected from nitrogen (N), oxygen (O) and sulfur (S), or

[0322] The alkylene group is further substituted with one or more alkyl groups having 1 to 20 carbon atoms.

[0323] Furthermore, in one embodiment of the present invention, the branched linkers each comprise a fifth unit, each active agent is bound to the fifth unit via a second linker, and the fifth unit is bound to the antibody via a first linker.

[0324] In one embodiment of the present invention, the fifth unit is an amide, and the first linker comprises a carbonyl group of the amide.

[0325] In one embodiment of the present invention, the fifth unit is a lysine unit.

[0326] Specifically, in one embodiment of the present invention, at least one carbon atom of the alkylene group is substituted with nitrogen, the first linker comprises at least two atoms of a hydrophilic amino acid, and the nitrogen forms a peptide bond together with the backbone carbonyl group of the hydrophilic amino acid. For example, the hydrophilic amino acid can be arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

[0327] In one embodiment of the present invention, the branched linker of the antibody-active agent is an amino acid having a side chain that carries a charge at neutral pH in aqueous solution, preferably comprising arginine, aspartic acid, glutamic acid, lysine, or ornithine. The amino acid can be located at any position in the branched linker. For example, the oxime of the branched linker can be covalently bonded to the polyethylene glycol unit of the branched linker. Alternatively or additionally, such an amino acid can be present in the second linker, and can be present in any of the second linkers.

[0328] Specifically, in one embodiment of the present invention, the linker-active agent comprises a compound with the following structure.

[0329] Right now, ,

[0330] The above-mentioned B' and B" represent active agents which may be the same or different.

[0331] n1 to n3 each independently represent an integer from 0 to 30,

[0332] AA represents an amino acid group.

[0333] In one embodiment of the present invention, the above-mentioned AA represents an amino acid group, and an amino acid group refers to a combination of more than one amino acids.

[0334] In one embodiment of the present invention, i) the linker comprises a peptide sequence of multiple amino acids, and ii) at least two active agents are covalently bonded to the side chains of the amino acids.

[0335] Specifically, in one embodiment of the present invention, the above-mentioned amino acid group is a main chain bond or side chain bond of 1 to 20 amino acids.

[0336] Specifically, in one embodiment of the present invention, the amino acid group is a group consisting of main chain bonds or side chain bonds of 1 to 20 arginine, aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine, ornithine, proline, serine or threonine.

[0337] Specifically, in one embodiment of the present invention, the above-mentioned amino acid group is a group consisting of main chain bonds or side chain bonds of 1 to 20 arginine, aspartic acid, glutamic acid, lysine or ornithine.

[0338] Specifically, in one embodiment of the present invention, the amino acid group comprises one or more lysines among 1 to 20 amino acids.

[0339] Furthermore, the amino acid group includes the main chain bond and side chain bond of lysine.

[0340] In one embodiment of the present invention, the branched linker comprising the fifth unit may be .

[0341] In this specification, "active agent moiety" refers to a compound comprising an active agent and covalently bonded to a linker or a portion of a linker.

[0342] In the present invention, the active agent moiety can be directly bonded to the linker. Specifically, one or more, two or more, three or more, or four or more active agent moieties can be directly bonded to the linker.

[0343] Furthermore, in the present invention, the active agent moiety may include a compound unit that directly bonds the active agent to the linker. Furthermore, in the present invention, a portion of the active agent moiety may be cleavable, allowing the active agent to be released into the intracellular environment. Furthermore, in the present invention, the number of bonds between the active agent moiety and the linker may be determined based on the DAR, pharmacological activity, and other factors of the conjugate. In one example, the active agent moiety may include the entire structure below and the active agent, thereby enabling direct bonding between the active agent and the linker. However, this is merely an example and is not intended to be limiting.

[0344]

[0345] (The definitions of each part are the same as above.)

[0346] In one embodiment of the invention, the active agents are each independently selected from chemotherapeutic agents and toxins.

[0347] Furthermore, the active agent may be an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof, and may be selected from the following active agents:

[0348] (a) Erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate mesylate), PTK787 / ZK222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimine nimine), altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolomelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 88), dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard mustard), carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore), aclacinomycins, actinomycin, antrmycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin,6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acidacid), nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiabendazole Thiamiprine, thiguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone, propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, enureaEniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate nitrate), hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide K, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid acid), triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, paclitaxel, albumin-engineered nanoparticle formulations of paclitaxelformulation of paclitaxel), docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, oxaliplatin (carboplatin), vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid acid), capecitabine or a pharmaceutically acceptable salt, solvate or acid thereof;

[0349] (b) monokine, lymphokine, traditional polypeptide hormone, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormone, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-α, tumor necrosis factor-β, mullerian inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor factor), thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor (CSF), macrophage-colony stimulating factor, granulocyte-macrophage-colony stimulating factor (granulocyte-macrophage-CSF), granulocyte-colony stimulating factor, interleukin (IL), interleukin-1, interleukin-1α, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, tumor necrosis factor, polypeptide factor, LIF, kit ligand (kit ligand) or their complexes;

[0350] (c) Diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitine, α-amanitine, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, indolebenzodiazepine, pyridobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamycin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analogs analog), auristatin, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotic, esperamicin, epothilone, toxoid, or complexes thereof;

[0351] (d) affinity ligand, wherein the affinity ligand is a substrate, inhibitor, initiator, neurotransmitter, radioisotope or a complex thereof;

[0352] (e) Radioactive labels, 32P, 35S, fluorescent dyes, electron dense reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acid molecules with a sequence complementary to a target, or complexes thereof;

[0353] (f) immunomodulatory compounds, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents and anti-parasitic agents or their complexes;

[0354] (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene;

[0355] (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole;

[0356] (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine;

[0357] (j) aromatase inhibitors;

[0358] (k) protein kinase inhibitors;

[0359] (l) lipid kinase inhibitors;

[0360] (m) antisense oligonucleotides;

[0361] (n) Ribozyme;

[0362] (o) vaccines; and

[0363] (p) Anti-angiogenic agent.

[0364] In one embodiment of the present invention, the active agent is

[0365] Wherein, y is an integer from 1 to 10.

[0366] Furthermore, in another embodiment of the present invention, the active agent is a pyrrolobenzodiazepine dimer, and the linker is connected to the antibody via the N10 position or N10' position of the pyrrolobenzodiazepine dimer.

[0367] At the N10 position and the N10' position of the pyrrolobenzodiazepine dimer, one group independently selected from the group consisting of -C(O)O-*, -S(O)O-*, -C(O)-*, -C(O)NR-*, -S(O)2NR-*, -(P(O)R')NR-*, -S(O)NR-* and -PO2NR-* is attached,

[0368] Where * is the part where the linker is attached,

[0369] wherein R and R' are each independently substituted by H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C1-8 alkoxy, substituted or unsubstituted C1-8 alkylthio, substituted or unsubstituted C3-20 heteroaryl, substituted or unsubstituted C5-20 aryl, or mono-C1-8 alkylamino or di-C1-8 alkylamino,

[0370] wherein, when C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, or C5-20 aryl is substituted, it is substituted by a substituent selected from the group consisting of H, OH, N3, CN, NO2, SH, NH2, ONH2, NNH2, halogen, C1-6 alkyl, C1-6 alkoxy, and C6-12 aryl,

[0371] A pyrrolobenzodiazepine dimer prodrug, a pharmaceutically acceptable salt or solvate thereof can be used as the active agent.

[0372] More specifically, the pyrrolobenzodiazepine dimer is substituted by X at the N10 position or by X' at the N'10 position, wherein X or X' enables the pyrrolobenzodiazepine dimer to be linked to the linker.

[0373] X and X' are each independently selected from -C(O)O*, -S(O)O-*, -C(O)-*, -C(O)NRX-*, -S(O)2NRX-*, -P(O)R'NRX-*, -S(O)NRX-* or -PO2NRX-*,

[0374] Rx and Rx' are each independently selected from H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl or mono-C1-8 alkylamino or bis-C1-8 alkylamino.

[0375] In one embodiment of the present invention, a pyrrolobenzodiazepine dimer precursor is provided as the active agent. When administered in the form of the precursor, upon exposure to the blood, it undergoes an additional reaction to convert into the active drug. This prevents the possibility of side effects caused by unintended degradation of the linker. Furthermore, compared to existing PBD drugs, this has the advantage of being more stable by reducing toxicity to normal cells.

[0376] Furthermore, in the case of preparing antibody-drug conjugates, when the antibody-drug conjugates are prepared by existing methods, there is a risk of generating undesirable structural drugs due to the high content of impurities and the attack of exposed imine groups by nucleophiles. In contrast, when the antibody-drug conjugates are prepared by the method of the present invention, they have the advantage of being easy to separate due to their relatively high purity, and furthermore, they exhibit further improved physical properties compared to existing PBDs or PBD dimers.

[0377] In one embodiment of the present invention, the pyrrolobenzodiazepine dimer precursor is a pyrrolobenzodiazepine dimer precursor having the following general formula X structure or general formula XI structure, or a pharmaceutically acceptable salt or solvate thereof:

[0378] Formula X

[0379]

[0380] Formula XI

[0381]

[0382] In the above general formula,

[0383] The dotted lines indicate the selective presence of double bonds between C1 and C2, between C2 and C3, between C'1 and C'2, or between C'2 and C'3.

[0384] RX1 and RX1' are each independently selected from H, OH, =O, =CH2, CN, Rm, ORm, =CH-Rm'=C(Rm')2, O-SO2-Rm, CO2Rm, CORm, halogen or dihalogen,

[0385] Rm' is selected from Rm, CO2Rm, CORm, CHO, CO2H and halogen,

[0386] Rm is each independently selected from the group consisting of C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl,

[0387] RX2, RX2', RX3, RX3', RX5 and RX5' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRm2, NO2 and halogen,

[0388] RX4 and RX4' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRm2, NO2, halogen, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C5-12 aryl, 5- to 7-membered heteroaryl, -CN, -NCO, -ORn, -OC(O)Rn, -OC(O)NRnRn', -OS(O)Rn, -OS(O)2Rn, -SRn, -S(O)Rn, -S(O)2Rn, -S(O)NRnRn', -S( O)2NRnRn', -OS(O)NRnRn', -OS(O)2NRnRn', -NRnRn', -NRnC(O)Ro, -NRnC(O)ORo, -NRnC(O)NRoRo', -NRnS(O)Ro, -NRnS(O)2Ro, -NRnS(O)NRoRo', -NRnS(O)2NRoRo', -C(O)Rn, -C(O)ORn and -C(O)NRnRn',

[0389] X and X' are each independently selected from -C(O)O*, -S(O)O-*, -C(O)-*, -C(O)NRX-*, -S(O)2NRX-*, -P(O)R'NRX-*, -S(O)NRX-* or -PO2NRX-*,

[0390] RX and RX' are each independently selected from H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl, or mono-C1-8 alkylamino or di-C1-8 alkylamino,

[0391] Y and Y' are each independently selected from O, S and N(H),

[0392] RX6 is independently selected from C3-12 alkylene, C3-12 alkenylene or C3-12 heteroalkylene,

[0393] RX7 and RX7' are each independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C6-10 aryl, 5-membered to 7-membered heteroaryl, -ORr, -OC(O)Rr, -OC(O)NRrRr', -OS(O)Rr, -OS(O)2Rr, -SRr, -S(O)Rr, -S(O)2Rr, -S(O)NRrRr', -S(O)2NRrRr', -OS(O)NRrRr', -OS(O)2NRrRr', -NRr Rr', -NRrC(O)Rs, -NRrC(O)ORs, -NRrC(O)NRsRs', -NRrS(O)Rs, -NRrS(O)2Rs, -NRrS(O)NRsRs', -NRrS(O)2NRsRs, -C(O)Rr, -C(O)ORs or -C(O)NRrRr',

[0394] Rr, Rr', Rs and Rs' are each independently selected from H, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C3-13 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C5-10 aryl and 5-membered to 7-membered heteroaryl,

[0395] Rx8 and Rx8' are each independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 heteroalkyl, 3- to 7-membered heterocycloalkyl, C5-10 aryl, 5- to 7-membered heteroaryl, -S(O)Rm, -S(O)2Rm, -S(O)NRmRm', -S(O)2NRmRm', -NRmRm', -NRmC(O)Rm, -NRmC(O)ORn, -NRmC(O)NRnRn', -NRmS(O)Rn, -NRmS(O)2Rn, -NRmS(O)NRnRn', -NRmS(O)2NRnRn', -C(O)Rm, -C(O)ORm and -C(O)NRmRm',

[0396] Za is selected from ORX12a, NRX12aRX12a or SRX12a,

[0397] Zb is selected from ORX13a, NRX13aRX13a or SRX13a,

[0398] Za' is selected from ORX12a', NRX12a', RX12a' or SRX12a',

[0399] Zb' is selected from ORX13a', NRX13'RX13a' or SRX13a',

[0400] RX12a, RX12a', RX13a' and RX13a' are each independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C5-10 aryl, 5- to 7-membered heteroaryl, -C(O)RX15a, -C(O)ORX15a and -C(O)NRX15aRX15a',

[0401] RX15a and RX15a' are each independently selected from C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl,

[0402] The above Za and Zb, together with the atoms to which they are attached, arbitrarily combine to form a 3-membered to 7-membered heterocyclic group, a 3-membered to 7-membered heterocycloalkyl group or a 3-membered to 7-membered heteroaryl group; Za' and Zb', together with the atoms to which they are attached, arbitrarily combine to form a 3-membered to 7-membered heterocyclic group, a 3-membered to 7-membered heterocycloalkyl group or a 3-membered to 7-membered heteroaryl group,

[0403] The above Rn, Rn', Ro, Ro', Rp and Rp' are each independently selected from H, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C3-13 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C5-10 aryl and 5-membered to 7-membered heteroaryl.

[0404] In one embodiment of the present invention, each of the above Rm is independently selected from the group consisting of C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-membered heterocyclyl, 3- to 7-membered heterocycloalkyl, and 5- to 7-membered heteroaryl.

[0405] In one embodiment of the present invention, Rm is further substituted by C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3-membered to 7-membered heterocyclyl, 3-membered to 7-membered heterocycloalkyl or 5-membered to 7-membered heteroaryl.

[0406] In one embodiment of the present invention, the above-mentioned Rx4 and Rx4' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRmRm', NO2, halogen, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C5-12 aryl, 5- to 7-membered heteroaryl, -CN, -NCO, -ORn, -OC(O)Rn, -OC(O)NRnRn', -OS(O)Rn, -OS(O)2Rn, -SRn, -S(O)Rn, -S(O)2Rn, -S(O)NRnRn', -S(O)2NRnRn', -OS(O)NRnRn', -OS(O)2NRnRn', -NRnRn', -NRnC(O)Ro, -NRnC(O)ORo, -NRnC(O)NRoRo', -NRnS(O)Ro, -NRnS(O)2Ro, -NRnS(O)NRoRo', -NRnS(O)2NRoRo', -C(O)Rn, -C(O)ORn and -C(O)NRnRn',

[0407] The above RX4 or RX4' is C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C5-12 aryl or 5-membered to 7-membered heteroaryl, and is further substituted by one or more C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C5-10 aryl, 5-membered to 7-membered heteroaryl, -ORp, -OC(O)Rp, -OC(O)NRpRp', -OS(O)Rp, -OS(O)2Rp, -SRp , -S(O)Rp, -S(O)2Rp, -S(O)NRpRp', -S(O)2NRpRp', -OS(O)NRpRp', -OS(O)2NRpRp', -NRpRp', -NRpC(O)Rq, -NRpC(O)ORq , -NRpC(O)NRqRq', -NRpS(O)Rq, -NRpS(O)2Rq, -NRpS(O)NRqRq', -NRpS(O)2NRqRq', -C(O)Rp, -C(O)ORp or -C(O)NRpRp substitution.

[0408] In one embodiment of the present invention, the above-mentioned Rx7 and Rx7' are each independently selected from H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3- to 7-membered heterocycloalkyl, C6-10 aryl, 5- to 7-membered heteroaryl, -ORr, -OC(O)Rr, -OC(O)NRrRr', -OS(O)Rr, -OS(O)2Rr, -SRr, -S(O)Rr, -S(O)2Rr, -S(O)NRrRr' , -S(O)2NRrRr', -OS(O)NRrRr', -OS(O)2NRrRr', -NRrRr', -NRrC(O)Rs, -NRrC(O)ORs, -NRrC(O)NRsR s', -NRrS(O)Rs, -NRrS(O)2Rs, -NRrS(O)NRsRs', -NRrS(O)2NRsRs, -C(O)Rr, -C(O)ORs or -C(O)NRrRr',

[0409] The above RX7 or RX7' is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C6-10 aryl, 5-membered to 7-membered heteroaryl, and is further replaced by C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-6 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C6-10 aryl, substituted with 5- to 7-membered heteroaryl, -ORt, -OC(O)Rt, -OC(O)NRtRt', -OS(O)Rt, -OS(O)2Rt, -SRt, -S(O)Rt, -S(O)2Rt, -S(O)NRtRt', -S(O)2NRtRt', -OS(O)NRtRt', -OS(O)2NRtRt', -NRtRt', -NRtC(O)Ru, -NRtC(O)ORu, -NRtC(O)NRuRu', -NRtS(O)Ru, -NRtS(O)2Ru, -NRtS(O)NRuRu', -NRtS(O)2NRuRu', -C(O)Rt, -C(O)ORt or -C(O)NRtRt',

[0410] The above-mentioned Rr, Rr', Rs, Rs', Rt, Rt', Ru and Ru' are each independently selected from H, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C3-13 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C5-10 aryl and 5-membered to 7-membered heteroaryl.

[0411] In one embodiment of the present invention, the above RX1 and RX1' are each independently selected from Rm,

[0412] Rm is selected from C1-6 alkyl, C2-6 alkenyl, C5-7 aryl and C3-6 heteroaryl.

[0413] In one embodiment of the present invention, RX2, RX2', RX3, RX3', RX5 and RX5' are each independently selected from H or OH.

[0414] In one embodiment of the present invention, RX4 and RX4' are each independently selected from Rm, and

[0415] Rm is a C1-6 alkoxy group.

[0416] In one embodiment of the present invention, RX4 and RX4' are each independently selected from the group consisting of methoxy, ethoxy and butoxy.

[0417] In one embodiment of the present invention, Y and Y' are 0.

[0418] In one embodiment of the present invention, the above-mentioned R X6 is C3-12 alkylene, C3-12 alkenylene or C3-12 heteroalkylene, and the above-mentioned R X6 is substituted by -NH2, -NHRm, -NHC(O)Rm, -NHC(O)CH2-[OCH2CH2]n-RXX or -[CH2CH2O]n-RXX,

[0419] The above RXX is H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl or mono-C1-8 alkylamino or di-C1-8 alkylamino,

[0420] n is an integer of 1 to 6.

[0421] In one embodiment of the present invention, the pyrrolobenzodiazepine dimer is represented by the following general formula X:

[0422] Formula X

[0423]

[0424] In the above general formula,

[0425] The dotted lines indicate the selective presence of double bonds between C1 and C2, between C2 and C3, between C'1 and C'2, or between C'2 and C'3.

[0426] RX1 and RX1' are each independently selected from H, OH, =O, =CH2, CN, Rm, ORm, =CH-Rm, =C(Rm')2, OSO2-Rm, CO2Rm, CORm, halogen or dihalogen,

[0427] RX2, RX2'RX3 and RX3' are each independently selected from H, Rm, OH, ORm, NRm2, NO2 and halogen,

[0428] RX4 and RX4' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRm2, halogen and C1-6 alkyl,

[0429] Rx5 and Rx5' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRm2, -NRmRm', NO2, -NRmC(O)Rm', -NRmC(O)ORm', -NRmC(O)NRmRm', -S(O)Rm, -S(O)2Rm, -S(O)NRmRm', -S(O)2NRmRm', -NRmS(O)Rm', -NRmS(O)2Rm', -P(O)Rm, -P(O)2Rm, -P(O)NRmRm', -P(O)2NRmRm', -NRmP(O)Rm', -NRmP(O)2Rm' and halogen,

[0430] Y and Y' are each independently O, S or N(H),

[0431] RX6 is independently C3-12 alkylene, C3-12 alkenylene or C3-12 heteroalkylene,

[0432] RX6 is substituted by -NH2, -NHRm, -NHC(O)Rm, -NHC(O)CH2-[OCH2CH2]n-RXX or -[CH2CH2O]n-RXX or is unsubstituted,

[0433] The above RXX is H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl or mono-C1-8 alkylamino or di-C1-8 alkylamino, wherein n is an integer from 1 to 6,

[0434] RX7 and RX7' are each independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -C(O)Rr, -C(O)ORs or -C(O)NRrRr',

[0435] Rr, Rr' and Rs are each independently H, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, C3-13 cycloalkyl, 3-membered to 7-membered heterocycloalkyl, C5-10 aryl or 5-membered to 7-membered heteroaryl,

[0436] Rm' is independently selected from Rm, CO2Rm, CORm, CHO, CO2H and halogen,

[0437] Rm is each independently selected from the group consisting of H, OH, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3-membered to 7-membered heterocyclyl, 3-membered to 7-membered heterocycloalkyl and 5-membered to 7-membered heteroaryl, wherein one or more hydrogen atoms of the C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3-membered to 7-membered heterocyclyl, 3-membered to 7-membered heterocycloalkyl and 5-membered to 7-membered heteroaryl may be substituted with OH, ═O, C1-12 alkyl or C1-12 alkoxy.

[0438] In one embodiment of the present invention, RX1 and RX1' are each independently selected from =CH2, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C5-7 aryl, C3-6 heteroaryl or C5-7 aryl substituted by C1-6 alkyl or C1-6 alkoxy.

[0439] In one embodiment of the present invention, RX2, RX2', RX3 and RX3' are each independently selected from H or OH.

[0440] In one embodiment of the present invention, RX5 and RX5' are each independently selected from the group consisting of H, OH, -S(O)Rm, S(O)2Rm, -P(O)Rm and -P(O)2Rm, wherein Rm is H, OH, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl or C2-12 alkynyl.

[0441] In one embodiment of the present invention, RX4 and RX4' are each independently a C1-6 alkoxy group.

[0442] In one embodiment of the present invention, RX4 and RX4' are each independently selected from the group consisting of methoxy, ethoxy and butoxy.

[0443] In one embodiment of the present invention, Y and Y' are 0.

[0444] In one embodiment of the present invention, the above-mentioned R X6 is C3-12 alkylene, C3-12 alkenylene or C3-12 heteroalkylene, and the above-mentioned R X6 is substituted by -NH2, -NHRm, -NHC(O)Rm, -NHC(O)CH2-[OCH2CH2]n-RXX or -[CH2CH2O]n-RXX,

[0445] The above RXX is H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl or mono-C1-8 alkylamino or di-C1-8 alkylamino,

[0446] n is an integer of 1 to 6.

[0447] Furthermore, in one embodiment of the present invention, the active agent is a pyrrolobenzodiazepine dimer, represented by the general formula XII or XIII:

[0448] Formula XII

[0449]

[0450] General formula XIII

[0451]

[0452] The above Xa and Xa' are independently selected from a bond or a C1-6 alkylene group,

[0453] ZX' and ZX are each independently selected from hydrogen, C1-8 alkyl, halogen, cyano, nitro, 、 or -(CH2)m-OCH3,

[0454] R80, R90 and R100 are each independently selected from hydrogen, C1-8 alkyl, C2-6 alkenyl and C1-6 alkoxy,

[0455] m is an integer from 0 to 12.

[0456] The above ZX' and ZX are each independently selected from hydrogen, 、 and (CH2)m-OCH3,

[0457] Each of R80, R90 and R100 is selected from the group consisting of hydrogen, C1-3 alkyl and C1-3 alkoxy.

[0458] m is an integer of 1 to 6.

[0459] In one embodiment of the present invention, the [LINKER-(B)1] precursor is selected from One of the groups composed of.

[0460] Furthermore, in one embodiment of the present invention, the [LINKER-(B)1] precursor is

[0461] Among them, MMAE is monomethyl auristatin E.

[0462] MMAF is monomethyl auristatin F.

[0463] In one embodiment of the present invention, the [LINKER-(B)1] precursor comprises the following structure:

[0464] The above-mentioned B' and B" represent active agents which may be the same or different.

[0465] m and n each independently represent an integer from 0 to 30.

[0466] Specifically, the pyrrolobenzodiazepine dimer refers to the portion connected to X and X' of the general formula X. More specifically, .

[0467] Furthermore, the present invention relates to a pharmaceutical composition comprising the above-mentioned conjugate for preventing or treating hyperproliferation, cancer or angiogenesis diseases.

[0468] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically effective amount of a chemotherapeutic agent.

[0469] In one embodiment of the present invention, the above-mentioned cancer is one selected from the group consisting of lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, intestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma and melanoma.

[0470] Furthermore, the present invention relates to a pharmaceutical preparation comprising the above-mentioned conjugate.

[0471] The conjugates of the present invention can be administered, for example, orally or parenterally. Parenteral administration refers to a broad range of routes of administration, including, for example, transdermal, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, sublingual, intrathecal, inhalation, ocular, rectal, vaginal, and intraventricular administration.

[0472] When preparing the above composition, it can be prepared by using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0473] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and lozenges. These solid preparations are prepared by adding at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, to one or more compounds of the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents of water and liquid paraffin, they may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives.

[0474] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, injections, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions for parenteral administration include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include semi-synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, glyceryl laurate, glycerin, and gelatin.

[0475] The conjugate of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to drug treatment. The effective dosage level can be determined based on factors including the type of disease of the patient, the severity of the disease, the drug activity, the drug sensitivity, the administration time, the administration route and excretion rate, the treatment time, the drugs used simultaneously, and other factors known in the medical field. The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered once or multiple times. Considering all the factors described above, it is particularly important to administer the drug in an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those of ordinary skill in the art to which the present invention pertains.

[0476] Specifically, the effective amount of the conjugate of the present invention may vary according to the age, sex, and weight of the patient. Generally, it is administered daily or every other day at a dosage of 0.01 mg to 150 mg / 1 kg body weight, and more specifically, at a dosage of 0.1 mg to 100 mg / 1 kg body weight, or administered 1 to 3 times a day. However, since the dosage may increase or decrease depending on the administration route, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way. Effect of the Invention

[0477]

[0478] The antibody-drug conjugate of the present invention comprises an anti-CLDN18.2 antibody that specifically binds to and internalizes into cells expressing claudin 18 isoform 2 (CLDN18.2). Thereby, the drug can be specifically and selectively effectively delivered, and the drug is stably bound to the antibody, so that it can maintain stability in vivo while exhibiting the desired cytotoxicity. In particular, it exhibits the effect of providing an antibody-linker-drug (toxin) system, which combines a linker technology. The linker contains a self-cleaving group that is more stable in plasma and stable even during in vivo circulation, and enables the drug to be easily released in cancer cells to maximize the drug efficacy, so that the drug and / or toxin can stably reach the target cells and can significantly reduce toxicity while effectively exerting the drug efficacy. Brief Explanation of Drawings

[0479] Figure 1 is a result diagram of a cell binding experiment using a flow cytometer to confirm the cancer cell binding ability of an antibody.

[0480] FIG2 and FIG3 are graphs showing the results of in vivo experiments on ADC samples.

[0481] 4 to 7 are schematic diagrams of the preparation process of the ADC of the present invention. Implementation Method

[0482] Hereinafter, the present invention will be further described in detail through embodiments and experimental examples.

[0483] The following examples and experimental examples are provided to help understand the present invention and are not intended to limit the scope of protection of the present invention.

[0484] Example 1. Preparation of claudin 18 isoform 2 (CLDN18.2)-specific antibodies

[0485] Antibodies that specifically bind to CLDN18.2 were prepared using the method described in International Patent No. PCT / CN2020 / 118650. The amino acid sequences of the antibodies that specifically bind to CLDN18.2 are shown in Table 1 below.

[0486] Table 1

[0487] To synthesize antibody-drug conjugates, we generated the antibody clone PR301839, which contains a CaaX peptide (GGGGGGGCVIM; SEQ ID NO: 19) fused to the C-terminus of the light chain (SEQ ID NO: 18) of the antibody listed in Table 1 (PR002726). The antibody was produced by transient expression in CHO cells. The resulting PR301839 antibody was used for the next round of antibody-drug conjugate synthesis.

[0488] Table 2

[0489] Example 2. Preparation of Compound 1 and Compound 2

[0490]

[0491]

[0492] The above-mentioned compounds 1 and 2 were prepared by the method described in Korean Patent Publication No. 10-2018-0110645.

[0493] Example 3. Preparation of Compound 3

[0494]

[0495] The above compound 3 was prepared by the method described in International Patent Publication No. WO2017-089895.

[0496] In the above compound 3, the structure of MMAE is shown below.

[0497]

[0498] Example 4. Preparation of Compound 4 and Compound 5

[0499]

[0500] The above compounds 4 and 5 were prepared by the method described in patent WO2017-089890.

[0501] In the above compound 5, the structure of MMAF is shown below.

[0502]

[0503] Example 5. Preparation of Antibody Drug Conjugates

[0504] The antibody-drug conjugate was prepared in two steps. LCB14-0606 and LCB14-0512 were prepared using the method described in Korean Patent Publication No. 10-2014-0035393. The structural formulas of LCB14-0606 and LCB14-0512 are shown below:

[0505]

[0506] Step 1: Preparation of prenylated antibodies

[0507] A prenylation reaction mixture of the antibody prepared in Example 1 was prepared and reacted at 30°C for 16 hours. The reaction mixture consisted of 24 μM antibody, 400 nM FTase (Calbiochem #344145), and 0.25 mM LCB14-0511 or LCB14-0606 in a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 10 μM ZnCl2, and 0.25 mM DTT). After the reaction, the prenylated antibody was desalted using a G25 Sepharose column (AKTA Purifier, GE Healthcare) equilibrated with PBS.

[0508] Step 2: Drug-conjugation method

[0509] Conjugation by oxime bond formation

[0510] The reaction mixture for oxime bond formation between the prenylated antibody and the linker-drug was prepared by mixing 100 mM sodium acetate buffer (pH 5.2), 10% DMSO, 20 μM antibody, and 200 μM linker-drug (in-house, Compounds 1, 2, 3, 4, 5, 7, and 8 from Examples 1 and 2) with gentle stirring at 30°C. After 6 or 24 hours of reaction, excess low-molecular-weight compounds were removed by FPLC (AKTA Purifier, GE Healthcare), and the concentrated protein fraction was collected.

[0511] Copper-free click conjugation reaction

[0512] For the antibody-drug conjugate generated by the copper-free click binding reaction between the isoprenylated antibody and the linker-drug, the reaction mixture was prepared by mixing PBS buffer (pH 7.4), 1% DMSO, 10 μM isoprenylated antibody and 100 μM linker-drug (internally, compound 2 of Example 1). After 6 hours of reaction at 25°C, excess low molecular weight compounds were removed by FPLC (AKTA purifier, GE healthcare) or G25 Sepharose column process and the concentrated protein fractions were collected.

[0513] Table 3

[0514] Antibody Drug Conjugate Preparation Catalog

[0515] Experimental Example 1. Evaluation of Antibody Binding Ability to Cancer Cells

[0516] The cancer cell binding ability of the anti-CLDN18.2 antibody (PR002726) and the antibody (PR301839) used in the preparation of an antibody-drug conjugate in which a CaaX peptide is fused to the C-terminus of the light chain were confirmed by cell binding experiments using flow cytometry (FACS).

[0517] As cancer cells, gastric cancer cell lines SNU-601 and SNU-620, and pancreatic cancer cell line PATU-8988s, known to express CLDN18.2, were used. As control antibodies, a group treated with human IgG and a group treated only with the secondary antibody were used.

[0518] The experimental results confirmed that in SNU-601, the binding abilities of PR002726 and PR301839 were 22.95-fold and 17.5-fold stronger, respectively, than those of the IgG control group. In SNU-620, the binding abilities of PR002726 and PR301839 were 15.09-fold and 11.21-fold stronger, respectively, than those of the control group. In PATU-8988s, the binding ability of PR301839 was 16.21-fold stronger than that of the control group. In summary, both PR002726 and PR301839 antibodies are capable of binding to cancer cells expressing CLDN18.2. (See Figure 1)

[0519] Experimental Example 2. In vitro cytotoxicity assessment

[0520] The cell proliferation inhibitory activity of the drugs and antibody-drug conjugates listed in Table 4 was measured against the cancer cell lines. The commercially available human gastric cancer cell lines NUGC-4, SNU-601, and SNU-602, and the pancreatic cancer cell line PATU-8988s were used as cancer cell lines, and HaCaT, Fa2N-4, HK-2, and HS738.st / Int were used as normal cell lines. MMAE, SG2057, and the antibody-drug conjugates listed in Table 3 were used as drugs. In a 96-well plate, 2,500 to 10,000 cells were seeded per well for each treatment group treated with each cancer cell line for 144 hours and cultured for 24 hours. The cancer cell lines were treated with the drugs and antibody-drug conjugates at concentrations ranging from 0.256 pM to 100 nM (5-fold serial dilutions), while the normal cell lines were treated with the drugs and antibody-drug conjugates at concentrations ranging from 2.56 pM to 1000 nM (5-fold serial dilutions). After 144 hours, the number of living cells was quantified using sulforhodamine B (SRB) dye, and the Hs738.st / Int cell line was quantified using Cell titer glo.

[0521] Table 4

[0522] Cytotoxicity assays with comparable reaction times confirmed that an auristatin-type antibody-drug conjugate (ADC3) exhibited equivalent or greater cytotoxicity than pyrrolobenzodiazepine-type antibody-drug conjugates (ADC1 and ADC2) in the NUGC-4 cell line. Furthermore, CLDN18.2 antibodies exhibited stronger binding. In the SNU-601 cell line, which exhibited the strongest cytotoxicity, pyrrolobenzodiazepine-type antibody-drug conjugates (ADC1 and ADC2) exhibited greater cytotoxicity than auristatin-type antibody-drug conjugates (ADC3, ADC4, and ADC5) in comparable reaction times. In the SNU-620 cell line, which exhibited strong antibody binding, no significant difference in anticancer efficacy was observed between the two types of antibody-drug conjugates. Comparison of cytotoxicity against three auristatin-type antibody-drug conjugates in the pancreatic cancer cell line PATU-8988 revealed that all three ADCs exhibited potent cytotoxicity.

[0523] In normal cell lines HaCaT, Fa2N-4, HK-2, and Hs738.st / Int, ADC1 to ADC5 showed no cytotoxicity within the effective dose range. The cytotoxicity was significantly reduced compared to when the toxin was administered alone or to cancer cell lines. This indicates that the ADCs of the present invention exhibit cancer cell-specific cytotoxicity.

[0524] Experimental Example 3. In vivo efficacy evaluation

[0525] After culturing the gastric cancer cell line SNU-601, 20,000 cells were mixed with 100 μl of Matrigel in 100 μl of PBS and transplanted into Balb / c-nude mice. When tumors reached 150 mm³ to 200 mm³ in size, the antibody-drug complexes listed in Tables 5 and 6 were administered intravenously.

[0526] Table 5

[0527] Comparison of in vivo efficacy of antibody-drug conjugate samples (first time)

[0528] Table 6

[0529] Comparison of in vivo efficacy of ADC samples (second time)

[0530] The results of the first experiment confirmed that ADC1, a pyrrolobenzodiazepine antibody-drug complex, exhibited tumor growth inhibition at doses of 0.2 mg / kg and 0.4 mg / kg, compared to the control group. ADC3, an auristatin antibody-drug complex, exhibited dose-dependent tumor growth inhibition at doses of 0.5 mg / kg, 1.0 mg / kg, and 2.0 mg / kg, compared to the control group. The results of the in vivo efficacy comparison experiment (first experiment) for the ADC samples are shown in Figure 2. The results of the second experiment confirmed that the tumor growth inhibition efficacy of the ADC3 1 mg / kg group was similar to that of the first experiment. ADC4 and ADC5 exhibited dose-dependent tumor growth inhibition at all doses. The results of the in vivo efficacy comparison experiment (second experiment) for the ADC samples are shown in Figure 3.

[0531] References

[0532] Duncan, AR, JM Woof, LJ Partridge, DR Burton, and G. Winter. 1988. Localization of the binding site for the human high-affinity Fc receptoron IgG. Nature 332:563-564.

[0533] Lund, J., G. Winter, PT Jones, JDPound, T. Tanaka, MR Walker, PJ Artymiuk, Y. Arata, DR Burton, R. Jefferis, and JMWoof. 1991. Human FcγRI and FcγRII interact with distinct but overlapping sites on human IgG. J. Immunol. 147:2657-2662.

[0534] Sarmay,G.,J.Lund,Z.Rozsnayay,J.Gergeley,and R.Jefferis.1992.Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity(ADCC)through different types of human Fc receptor.Mol.Immunol.29:633-639.

Claims

1. A coupling compound or a pharmaceutically acceptable salt thereof, wherein, Represented by the following general formula IIa: General formula IIa, wherein Ab is an antibody or its antigen-binding fragment that specifically binds to tight junction protein 18 subtype 2 (CLDN18.2), the Ab comprising a heavy chain CDR1 composed of the amino acid sequence represented by sequence number 2, a heavy chain CDR2 composed of the amino acid sequence represented by sequence number 4, a heavy chain CDR3 composed of the amino acid sequence represented by sequence number 6, and a light chain CDR1 composed of the amino acid sequence represented by sequence number 9, a light chain CDR2 composed of the amino acid sequence represented by sequence number 11, and a light chain CDR3 composed of the amino acid sequence represented by sequence number 13; B is an active agent independently; And l and m are each independently an integer selected from 1 to 20; G is each independently; R3 is a hydrogen or carboxyl protecting group, R4 is each independently a hydrogen or hydroxyl protecting group, R1 and R2 are each independently hydrogen, C1-8 alkyl or C3-8 cycloalkyl, W is each independently -C(O)-, -C(O)NR'-, -C(O)O-, -SO2NR'-, -P(O)R''NR'-, -SONR'- or -PO2NR'-, wherein C, S or P are directly bonded to the benzene ring, and NR' is bonded to L; R' and R'' are each independently hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamine or bis-C1-8 alkylamine, C3-20 heteroaryl or C6-20 aryl; Z is independently C1-8 alkyl, hydrogen, halogen, cyano, or nitro; n is an integer from 0 to 3; and L independently comprises: A) a C1-50 alkylene or a 1-50 heteroalkylene comprising: (i) one or more unsaturated bonds; (ii) a heteroarylene; or (iii) a C1-20 alkyl substitution on the C1-50 alkylene; or B) comprising at least one isoprene unit recognizable by isoprene-like transferases, having a structure represented by the following general formula III: General formula III.

2. The conjugate as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, This Ab contains the variable heavy chain FR of a human antibody, encoded by a gene derived from germline V gene IGHV3-23.

3. The conjugate as described in claim 2 or a pharmaceutically acceptable salt thereof, wherein, The variable heavy chain FR comprises: heavy chain FR1 consisting of the amino acid sequence represented by serial number 1; heavy chain FR2 consisting of the amino acid sequence represented by serial number 3; heavy chain FR3 consisting of the amino acid sequence represented by serial number 5; and heavy chain FR4 consisting of the amino acid sequence represented by serial number 7.

4. The conjugate as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, This Ab contains the variable light chain FR of a human antibody, encoded by a gene derived from germline V gene IGKV3-11 or germline V gene IGKV3-15.

5. The conjugate as described in claim 4 or a pharmaceutically acceptable salt thereof, wherein, The variable light chain FR comprises: light chain FR1 consisting of an amino acid sequence represented by serial number 8; light chain FR2 consisting of an amino acid sequence represented by serial number 10; light chain FR3 consisting of an amino acid sequence represented by serial number 12; and light chain FR4 consisting of an amino acid sequence represented by serial number 14.

6. The conjugate as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, The Ab comprises: an amino acid sequence represented by Serial No. 15 or a heavy chain variable region consisting of an amino acid sequence having at least 80% sequence identity with it; and a light chain variable region consisting of an amino acid sequence represented by Serial No. 16 or an amino acid sequence having at least 80% sequence identity with it.

7. The conjugate as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein, The Ab can be a monoclonal antibody, a single-chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single-chain variable antibody (scFv), a scFv-Fc fragment, an antibody variant, a polyantibody, a microantibody, a double-chain antibody, a bispecific antibody, or a multispecific antibody.

8. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, G is each hydrogen, and R3 and R4 are each hydrogen.

9. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, R1 and R2 are both hydrogen, n is 0, W is independently -C(O)NR'-, C is directly bonded to the benzene ring, R' is hydrogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, mono-C1-8 alkylamine or bis-C1-8 alkylamine, C3-20 heteroaryl or C6-20 aryl, and NR' is bonded to L.

10. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L is a nitrogen-containing 1-50-membered heteroalkylene group, and L contains two or more atoms of a hydrophilic amino acid, which forms a peptide bond with the carbonyl group of the hydrophilic amino acid.

11. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L is covalently bonded to Ab via a thioether bond, which contains the sulfur atom of the cysteine ​​in Ab.

12. The conjugate as described in claim 11 or a pharmaceutically acceptable salt thereof, wherein, The C-terminus of the Ab contains an amino acid motif recognized by this type of isoprene transferase, and the thioether bond contains the sulfur atom of the cysteine ​​in the amino acid motif.

13. The conjugate as described in claim 12 or a pharmaceutically acceptable salt thereof, wherein, The amino acid motif is a CYYX sequence, where C is cysteine, Y are aliphatic amino acids, and X is selected from glutamine, glutamic acid, serine, cysteine, methionine, alanine, and leucine.

14. The conjugate as claimed in claim 13 or a pharmaceutically acceptable salt thereof, wherein Y is each independently selected from alanine, isoleucine, leucine, methionine and valine, or the amino acid motif has a CVIM sequence or a CVLL sequence.

15. The conjugate as described in claim 12 or a pharmaceutically acceptable salt thereof, wherein, At least one of the 1 to 20 amino acids preceding the amino acid motif is glycine.

16. The conjugate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 7, wherein the Ab contains the amino acid sequence GGGGGGGCVIM at its C-terminus.

17. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L is a 3- to 50-membered heteroalkylene group containing an oxime, and (1) the oxygen atom of the oxime is located on the side of L connected to W, and the carbon atom of the oxime is located on the side of L connected to Ab, or, (2) the carbon atom of the oxime is located on the side of L connected to W, and the oxygen atom of the oxime is located on the side of L connected to Ab.

18. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L contains an oxime and at least one isoprene unit covalently bonded to the oxime and Ab.

19. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L contains a second unit, represented by the following general formula VIII or general formula IX: General formula VIII -(CH2)r(V(CH2)p)q- General formula IX -(CH2CH2X)w- The V is a single bond, -O-, -S-, -NR21-, -C(O)NR22-, -NR23C(O)-, -NR24SO2- or -SO2NR25-, X is -O-, C1-8 alkylene or -NR21-, R21 to R25 are each independently hydrogen, C1-6 alkyl, C1-6 alkylC6-20 aryl or C1-6 alkylC3-20 heteroaryl, r is an integer from 0 to 10, p is an integer from 0 to 10, q is an integer from 1 to 20, and w is an integer from 1 to 20.

20. The conjugate as described in claim 19 or a pharmaceutically acceptable salt thereof, wherein, q is an integer from 1 to 10, r and p are 1 or 2 respectively, and V is -0-.

21. The conjugate as described in claim 19 or a pharmaceutically acceptable salt thereof, wherein, X is -O-, and w is an integer from 1 to 10.

22. The conjugate as described in claim 19 or a pharmaceutically acceptable salt thereof, wherein, L contains at least one polyethylene glycol monomer represented by or.

23. The conjugate as described in claim 19 or a pharmaceutically acceptable salt thereof, wherein, L contains an oxime, wherein at least one polyethylene glycol monomer causes the oxime to be covalently bonded to the surfactant.

24. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L contains a third unit, which is formed by the reaction of alkynes with azides, or by the reaction of aldehydes or ketones with hydrazine or hydroxylamine.

25. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L contains a third unit, which is represented by general formulas IVa, IVb, IVc, IVd, or IVe: General formula IVa, General formula IVb, General formula IVc, General formula IVd, General formula IVe, where, L1 and L2 are each independently a single bond or a C1-30 alkylene bond, and R11 is hydrogen or a C1-10 alkyl group.

26. The conjugate as described in claim 25 or a pharmaceutically acceptable salt thereof, wherein, L1 and L2 are each independently a single bond or a C11 alkylene or a C12 alkylene.

27. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, This type of isoprene transferase is either farnesyltransferase (FTase) or geraniolyltransferase (GGTase).

28. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, L comprises one or more branched linkers covalently bonded to Ab, wherein each branched linker comprises: i) a fifth unit covalently bonded to Ab via a first linker PL; ii) a first branch B1 in which a first active agent is covalently bonded to the fifth unit via a second linker SL and a cleaving group CG; iii) a second branch B2 in which a) a second active agent is covalently bonded to the fifth unit via a second linker SL and a cleaving group CG; or, b) a polyethylene glycol portion covalently bonded to the fifth unit, and each cleaving group being hydrolyzed to release the active agent from the coupling.

29. The conjugate as described in claim 28 or a pharmaceutically acceptable salt thereof, wherein, The fifth unit is , or , L2, L3 and L4 are each independently a direct bond or -CnH2n-, where n is an integer from 1 to 30, G1, G2 and G3 are each independently a direct bond, , , or , R30 is hydrogen or C1-30 alkyl, R40 is hydrogen or L5-COOR6, L5 is a direct bond or -Cn'H2n'-, where n' is an integer from 1 to 10, and R6 is hydrogen or C1-30 alkyl.

30. The conjugate as described in claim 28 or a pharmaceutically acceptable salt thereof, wherein, This cleaving group can cleave within the target cell or release more than one active agent.

31. The conjugate as described in claim 28 or a pharmaceutically acceptable salt thereof, wherein, The conjugate contains one, two, three, four or more branched linkers, and each branched linker is bound to one or more active agents.

32. The conjugate as described in claim 28 or a pharmaceutically acceptable salt thereof, wherein, The fifth unit is lysine, and the first linker is connected to the C-terminus of the lysine.

33. The conjugate as described in claim 28 or a pharmaceutically acceptable salt thereof, wherein, The conjugate comprises a structure represented by the following: , B' and B'' represent active agents that can be the same or different, n1 to n3 are each an independent integer from 0 to 30, and AA represents an amino acid group.

34. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, The active agent is a chemotherapeutic agent or a toxin.

35. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, The active agent is an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

36. The conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, The active agents are: (a) erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, and imatinib mesylate. mesylate), PTK787 / ZK222584, oxaliplatin, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, ethylenimine altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 88) Duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil Mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin AA) Clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, antimycin, azaserine, bleomycins, cactinomycin C, carrubicin, carminomycin, carzinophilin, chromophore Chromycins, Dactinomycin, Detorubucin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, Morpholino-Doxorubicin, Cyanomorpholino-Doxorubicin, 2-Pyrrolino-Doxorubucin, Liposome Doxorubicin, Deoxydoxorubicin, Epirubicin, Esorubicin, Marcellomycin, Mitomycin C, Mycophenolic Acidacid), nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamethoxam Purines, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, calusterone, dromostanolone, propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, demecolcine, diaziquone, elfornithine, elliptiniumacetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidamol, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharidek, razoxane, sizofiran, spirogermanium, tenuazonic acid acid), triaziquone, 2,2',2''-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactalol, pipobroman, arabinoside, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel.(a) paclitaxel, docetaxel, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, vincristine, vinorelbine, teniposide, edatrexate, daunomycin, aminopterin, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoic acid, capecitabine, or their pharmaceutically acceptable salts, solvates, or acids; (b) Monokines, lymphokines, polypeptide hormones, parathyroid hormone, thyroxine, relaxin, pro-relaxin, glycoprotein hormones, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone, liver growth factor, fibroblast growth factor, prolactin, placental prolactin, tumor necrosis factor, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinducible factor, interferon, colony-stimulating factor (CSF), macrophage-colony-stimulating factor, granulocyte-macrophage-colony-stimulating factor, granulocyte-colony-stimulating factor, interleukin (IL), polypeptide factors, LIF, kit ligands, or combinations thereof; (c) Diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, muscarinic acid, pyrrolobenzodiazepine, indobenzobenzodiazepine, pyridobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulolysin, geldanamycin, maytansinoid, calicheamycin, SG2285, dolastatin, cryptophycin, rhizoxin, CC-1065, enediyne antibiotics, esperamicin, epothilone, toxoids or combinations thereof; (d) Affinity ligands, wherein the affinity ligands are neurotransmitters, radioactive isotopes, or combinations thereof; (e)Radiolabeled substances, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acid molecules having a sequence complementary to the target, or combinations thereof; (f) immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or combinations thereof; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, LY117018, onapristone, or toremifene; (h) 4(5)-imidazole, megestrol acetate, exemestane, or anastrozole; (i) Flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (j) aromatase inhibitors; (k) protein kinase inhibitors; (l) lipid kinase inhibitors; (m) antisense oligonucleotides; (n) ribozymes; (o) vaccines; or (p) antiangiogenic agents.

37. The conjugate as described in claim 36 or a pharmaceutically acceptable salt thereof, wherein: (i) The tumor necrosis factor comprises tumor necrosis factor-α or tumor necrosis factor-β; (ii) The interferon comprises interferon-α, interferon-β or interferon-γ; (iii) The interleukin comprises interleukin-1, interleukin-1α, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11 or interleukin-12; (iv) The amanitin comprises α-amanitin; and (v) The radiolabel comprises 32P or 35S.

38. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, The active agent is a pyrrolobenzodiazepine dimer, which is substituted with X at the N10 position or with X' at the N'10 position, wherein X or X' causes the pyrrolobenzodiazepine dimer to be linked to the coupling agent. X and X' are each independently -C(O)O-*- or -C(O)-*, where * is the binding site between the pyrrolobenzodiazepine dimer and the linker.

39. A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, wherein, The active agent is represented by the general formula X: General formula X, where the dashed lines each represent double bonds as desired, G' is glucuronidyl or galactosidyl, Xa is independently selected from C1-6 alkylene groups, ZX' is independently selected from C1-8 alkyl, halogen, cyano, nitro, and –(CH2)m-OCH3, n is an integer from 1 to 3, R80 and R90 are each independently selected from hydrogen, C1-3 alkyl, C1-3 alkoxy and methoxyethyl, R100 is selected from hydrogen, C1-3 alkyl and C1-3 alkoxy, m is an integer from 1 to 12, X' is independently selected from -C(O)O-, -S(O)O-, -C(O)-, -C(O)NRX-, -S(O)2NRX-, -P(O)R'NRX-, -S(O)NRX- and -PO2NRX-, RX and RX' are each independently selected from H, OH, and C1-8 alkyl groups; RX1 and RX1' are each independently selected from H, OH, =O, =CH2, CN, Rm, ORm, =CH-Rm', =C(Rm')2, OSO2-Rm, CO2Rm, CORm, halogens, or dihalogens; RX2, RX2', RX3, and RX3' are each independently selected from H, Rm, OH, ORm, NRm2, NO2, and halogens; RX4 and RX4' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRm2, halogens, and C1-6 alkyl groups. RX5 and RX5' are each independently selected from H, Rm, OH, ORm, SH, SRm, NH2, NHRm, NRm2, -NRmRm', NO2, -NRmC(O)Rm', -NRmC(O)ORm', -NRmC(O)NRmRm', -S(O)Rm, -S(O)2Rm, -S(O)NRmRm', -S(O)2NRmRm', -NRmS(O)Rm', -NRmS(O)2Rm', -P(O)Rm, -P(O)2Rm, -P(O)NRmRm', -P(O)2NRmRm', -NRmP(O)Rm', -NRmP(O)2Rm', and halogens, and Y and Y' are each independently selected from O, S, or N(H). RX6 is independently a C3-12 alkylene, C3-12 alkenylene, or C3-12 heteroalkylene, and may be substituted or unsubstituted by -NH2, -NHRm, -NHC(O)Rm, -NHC(O)CH2-[OCH2CH2]n'-RXX, or -[CH2CH2O]n'-RXX, wherein RXX is H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl, or mono-C1-8 alkylamine or bis-C1-8 alkylamine, and n' is an integer from 1 to 6.RX7 and RX7' are each independently H, C1-6 alkyl, C2-6 alkenyl, C2-6 ynyl, -C(O)Rr, -C(O)ORs, or -C(O)NRrRr'; Rr, Rr', and Rs are each independently H, C1-7 alkyl, C2-7 alkenyl, C2-7 ynyl, C3-13 cycloalkyl, 3- to 7-membered heterocyclic alkyl, C5-10 aryl, or 5- to 7-membered heteroaryl; and Rm' is each independently selected from Rm, CO2Rm, CORm, CHO, CO2H, and halogens. Rm is independently selected from the group consisting of H, OH, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl, C2-12 alkynyl, C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-heterocyclic, 3- to 7-heterocyclic alkyl, and 5- to 7-heteroaryl, wherein at least one hydrogen atom of the C5-20 aryl, C5-20 heteroaryl, C3-6 cycloalkyl, 3- to 7-heterocyclic, 3- to 7-heterocyclic alkyl, and 5- to 7-heteroaryl can be substituted by OH, =O, C1-12 alkyl, or C1-12 alkoxy.

40. The conjugate as described in claim 39 or a pharmaceutically acceptable salt thereof, wherein, RX1 and RX1' are each independently =CH2, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C5-7 aryl, C3-6 heteroaryl, or C5-7 aryl substituted with C1-6 alkyl or C1-6 alkoxy.

41. The conjugate as described in claim 39 or a pharmaceutically acceptable salt thereof, wherein, RX2, RX2', RX3 and RX3' are each independently H or OH.

42. The conjugate as described in claim 39 or a pharmaceutically acceptable salt thereof, wherein, RX5 and RX5' are each independently selected from H, OH, -S(O)Rm, S(O)2Rm, -P(O)Rm and -P(O)2Rm, wherein Rm is H, OH, C1-12 alkyl, C1-12 alkoxy, C2-12 alkenyl or C2-12 alkynyl.

43. The conjugate as described in claim 39 or a pharmaceutically acceptable salt thereof, wherein, RX4 and RX4' are each independently C1-6 alkoxy groups.

44. The conjugate as described in claim 39 or a pharmaceutically acceptable salt thereof, wherein, Y and Y' are each independently O.

45. The conjugate as described in claim 39 or a pharmaceutically acceptable salt thereof, wherein, RX6 is a C3-12 alkylene, C3-12 alkenyl, or C3-12 heteroalkylene, and RX6 is substituted with -NH2, -NHRm, -NHC(O)Rm, -NHC(O)CH2-[OCH2CH2]n'-RXX or -[CH2CH2O]n'-RXX, where RXX is H, OH, N3, CN, NO2, SH, NH2, ONH2, NHNH2, halogen, C1-8 alkyl, C3-8 cycloalkyl, C1-8 alkoxy, C1-8 alkylthio, C3-20 heteroaryl, C5-20 aryl, or mono-C1-8 alkylamine or bis-C1-8 alkylamine, and n' is an integer from 1 to 6.

46. ​​A conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, comprising: , ... MMAE stands for monomethylolpropionate E, and MMAF stands for monomethylolpropionate F.

47. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 46 or a pharmaceutically acceptable salt thereof and an excipient.

48. The pharmaceutical composition as claimed in claim 47, further comprising a chemotherapeutic agent.

49. Use of a conjugate as described in any one of claims 1 to 46 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of diseases of excessive proliferation, cancer or angiogenesis.

50. The use as described in claim 49, wherein, This conjugate is used for the prevention or treatment of cancer, wherein the cancer is selected from lung cancer, gastrointestinal cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma.

51. The use as described in claim 50, wherein the lung cancer is small cell lung cancer.

52. The use as described in claim 50, wherein the gastrointestinal cancer is colorectal cancer.

53. A pharmaceutical preparation, wherein, The pharmaceutical formulation comprises any of the conjugates described in claims 1 to 46 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof, and is selected from injections, tablets, pills, powders, granules, capsules, lozenges, suspensions, oral solutions, emulsions, syrups, oils, lyophilized formulations and suppositories.

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