Antibody-drug conjugates and their use

Abstract

The present disclosure relates to antibody-drug conjugates and their use. Specifically, there is provided an antibody-drug conjugate represented by Formula I, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof. Ab-(L-D) a Formula I

Description

Cross - reference to related applications

[0001] This disclosure claims priority based on Chinese Patent Application No. 202210889771.5 filed on July 27, 2022, and the disclosure of the Chinese patent application is incorporated herein by reference in its entirety.

Technical Field

[0002] This disclosure relates to the field of pharmaceutical technology, and specifically, to antibody - drug conjugates and their use.

Background Art

[0003] Antibody - drug conjugates (ADCs) are a new type of therapeutic macromolecule that combines the powerful killing power of small - molecule drugs and the highly specific targeting of antibodies. By utilizing the specificity of antibodies, drugs can be accurately delivered to tumor sites or / and tumor cells, avoiding the death of normal cells in the body, and as a result, they can be used to reduce adverse reactions during the treatment process. An ADC consists of three components: an antibody, a drug, and a linker. When the antibody of the ADC specifically binds to a specific antigen on the surface of tumor cells, the cell membrane of the tumor cells caves inward, forming an endosome that engulfs the ADC. This process is also known as internalization. The antibody or linker of the ADC is decomposed by some enzymes inside the cell, thus releasing the drug, and then the released drug begins to act to kill the tumor cells. B7-H3 (B7 homolog 3 protein), also known as CD276 or B7RP-2, is a type of immune checkpoint molecule and belongs to the B7 family of immunomodulatory proteins. B7-H3 is lowly expressed in normal tissues but highly expressed in various solid tumors such as lung cancer, head and neck cancer, esophageal cancer, ovarian cancer, breast cancer, prostate cancer, melanoma, etc., and is associated with poor prognosis in a wide range of tumors. The use of B7-H3 as a therapeutic target could be an effective strategy for the treatment of cancers with high B7-H3 expression. Various B7-H3 targeted therapies, including monoclonal antibodies, bispecific antibodies, CAR-T, antibody-drug conjugates (ADCs), etc., are currently under development, but there are no approved drugs on the market yet. Among them, antibody-drug conjugates bring small molecule drugs (payloads) to target highly expressed tumor cells through specific antibodies against B7-H3, selectively reduce the off-target toxicity of small molecule drugs while retaining the tumor-killing properties of small molecule drugs, and have a greater potential to improve the benefit-risk ratio of anti-tumor therapies. This is expected to bring greater benefits to patients. Summary of the Invention Means for Solving the Problems

[0004] The present disclosure provides an antibody-drug conjugate represented by Formula I, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates. The antibody-drug conjugate exhibits excellent inhibitory activity against multiple tumor cell lines with different target expression levels and shows a strong in vivo growth inhibitory effect in a subcutaneous xenograft tumor model of human-derived tumor cells in nude mice. These indicate that great expectations can be placed on their applications. For this purpose, in a first aspect of the present disclosure, the present disclosure provides an antibody-drug conjugate represented by Formula I, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates. Ab-(L-D) a Formula I

[0005] Here, Ab is an antibody such as a monoclonal antibody or an antigen-binding fragment thereof; L is L 1 -L 2 -L 3 -L 4 ;

Chemical formula

Chemical formula

Chemical formula

[0006]

Chemical formula

[0007]

Chemical formula

[0008] In some embodiments, L 3is a peptide residue consisting of 2 to 4 (preferably 4) amino acid residues, with the amino terminus bonded to L 2 and the carbonyl terminus bonded to L 4 Preferably, the amino acids are selected from the group consisting of glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid, and aspartic acid. More preferably, the amino acids are selected from the group consisting of glycine and phenylalanine. In some embodiments, L 3 is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), with the amino terminus connected to L 2 and the carbonyl terminus connected to L 4

Chemical formula

[0009] In some embodiments, R 1 is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, R 1 is selected from the group consisting of hydrogen, halogen, and C1-C4 alkyl. In some embodiments, R 1 is C1-C6 alkyl. In some embodiments, R 1 is C1-C4 alkyl. In some embodiments, R1 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In some embodiments, R 1 is selected from the group consisting of methyl, ethyl, and n-propyl. In some embodiments, R 1 is selected from the group consisting of fluorine, chlorine, bromine, and iodine. In some embodiments, R 1 is selected from the group consisting of fluorine, chlorine, and bromine. ​In some embodiments, R 1 is methyl. In some embodiments, R 2 is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, R 2 is selected from the group consisting of hydrogen, halogen, and C1-C4 alkyl. In some embodiments, R 2 is C1-C6 alkyl. In some embodiments, R 2 is C1-C4 alkyl. In some embodiments, R 2 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In some embodiments, R2 is selected from the group consisting of methyl, ethyl, and n-propyl. In some embodiments, R 2 is halogen. In some embodiments, R 2 is selected from the group consisting of fluorine, chlorine, bromine, and iodine. In some embodiments, R 2 is selected from the group consisting of fluorine, chlorine, and bromine. In some embodiments, R 2 is fluorine. In some embodiments, R 2 is methyl.

[0010] In some embodiments, R 3 is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl. In some embodiments, R 3 is selected from the group consisting of hydrogen, halogen, and C1-C4 alkyl. In some embodiments, R 3 is selected from the group consisting of fluorine, chlorine, bromine, and iodine. In some embodiments, R 3 is selected from the group consisting of fluorine, chlorine, and bromine. In some embodiments, R 3is C1-C6 alkyl. In some embodiments, R 3 is C1-C4 alkyl. In some embodiments, R 3 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In some embodiments, R 3 is selected from the group consisting of methyl, ethyl, and n-propyl. In some embodiments, R 3 is methyl.

[0011] In some embodiments, L is

Chemical formula

[0012]

Chemical formula

Chemical formula

[0013] In some embodiments, the antibody-drug conjugate represented by Formula I is selected from the group consisting of the structural formulas in the following table.

Table 1a

[0014] In some embodiments, Ab is an anti - B7 - H3 antibody or an antigen - binding fragment thereof. In some embodiments, the anti - B7 - H3 antibody or an antigen - binding fragment thereof (a) The following three heavy - chain variable region (VH) complementarity - determining regions (CDRs): (i) The CDR1 sequence contained in VH shown in SEQ ID NO: 8, or a VH CDR1 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of 1 or 2 amino acids) as compared with the CDR1 sequence contained in said VH (ii) The CDR2 sequence contained in VH shown in SEQ ID NO: 8, or a VH CDR2 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of 1 or 2 amino acids) as compared with the CDR2 sequence contained in said VH; and (iii) The CDR3 sequence contained in VH shown in SEQ ID NO: 8, or a VH CDR3 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of 1 or 2 amino acids) with respect to the CDR3 sequence contained in said VH; and / or (b) The following three light chain variable region (VL) CDRs described below: (iv) The CDR1 sequence contained in VL shown in SEQ ID NO: 12, or a VL CDR1 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of 1 or 2 amino acids) with respect to the CDR1 sequence contained in said VL; (v) The CDR2 sequence contained in VL shown in SEQ ID NO: 12, or a VL CDR2 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of 1 or 2 amino acids) as compared with the CDR2 sequence contained in said VL; and (vi) The CDR3 sequence contained in VL shown in SEQ ID NO: 12, or a VL CDR3 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of 1 or 2 amino acids) with respect to the CDR3 sequence contained in said VL. In some embodiments, the substitution (or substituted) described in any one of (i) to (vi) is a conservative substitution.

[0015] In some embodiments, CDR1, CDR2, and CDR3 included in the heavy chain variable region (VH), and / or CDR1, CDR2, and CDR3 included in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system. In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the anti-B7-H3 antibody or an antigen-binding fragment thereof are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the anti-B7-H3 antibody or an antigen-binding fragment thereof are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. In some embodiments, the CDRs of the present disclosure are defined by the Kabat method. In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-B7-H3 antibody or an antigen-binding fragment thereof is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region of the anti-B7-H3 antibody or an antigen-binding fragment thereof is shown in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown in SEQ ID NO: 2. In a second aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the above antibody-drug conjugate, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates, and optionally one or more pharmaceutical excipients. In some embodiments, the antibody-drug conjugate, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates are present in an effective amount.

[0016] In a third aspect of the present disclosure, the present disclosure provides the use of the above antibody-drug conjugate, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates, or the above pharmaceutical composition, in the manufacture of a medicament for the treatment and / or prevention of a disease. Here, the disease is a disease related to abnormal B7-H3 expression or function. In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the disease is selected from the group consisting of lung cancer (e.g., undifferentiated lung cancer, small cell lung cancer, non-small cell lung cancer), esophageal cancer, gastric cancer, liver cancer, melanoma, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, thyroid cancer, sarcoma, cholangiocarcinoma, glioblastoma, and neuroblastoma. In a fourth aspect of the present disclosure, the present disclosure provides the above antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the above pharmaceutical composition for use in the treatment and / or prevention of a disease, wherein the disease is a disease associated with abnormal B7-H3 expression or function. In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the disease is selected from the group consisting of lung cancer (e.g., undifferentiated lung cancer, small cell lung cancer, non-small cell lung cancer), esophageal cancer, gastric cancer, liver cancer, melanoma, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, thyroid cancer, sarcoma, cholangiocarcinoma, glioblastoma, and neuroblastoma. In a fifth aspect of the present disclosure, the present disclosure provides a method for treating and / or preventing a disease, comprising administering to an individual in need thereof an effective amount of the above antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the above pharmaceutical composition, wherein the disease is a disease associated with abnormal B7-H3 expression or function. In some embodiments, the disease is cancer or an autoimmune disease.

[0017] In some embodiments, the disease is selected from the group consisting of lung cancer (e.g., undifferentiated lung cancer, small cell lung cancer, non-small cell lung cancer), esophageal cancer, gastric cancer, liver cancer, melanoma, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, thyroid cancer, sarcoma, cholangiocarcinoma, glioblastoma, and neuroblastoma. In a sixth aspect of the present disclosure, the present disclosure provides a composition for treating and / or preventing a disease, comprising the antibody-drug conjugate, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates. In some embodiments, the disease is a disease associated with abnormal B7-H3 expression or function. In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the disease is selected from the group consisting of lung cancer (e.g., undifferentiated lung cancer, small cell lung cancer, non-small cell lung cancer), esophageal cancer, gastric cancer, liver cancer, melanoma, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, thyroid cancer, sarcoma, cholangiocarcinoma, glioblastoma, and neuroblastoma. In a seventh aspect of the present disclosure, the present disclosure provides a medicament for treating and / or preventing a disease, comprising the antibody-drug conjugate, its stereoisomers, its prodrugs, its pharmaceutically acceptable salts, or its pharmaceutically acceptable solvates as an active ingredient. In some embodiments, the disease is a disease associated with abnormal B7-H3 expression or function. In some embodiments, the disease is cancer or an autoimmune disease. In some embodiments, the disease is selected from the group consisting of lung cancer (e.g., undifferentiated lung cancer, small cell lung cancer, non-small cell lung cancer), esophageal cancer, gastric cancer, liver cancer, melanoma, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, thyroid cancer, sarcoma, cholangiocarcinoma, glioblastoma, and neuroblastoma.

[0018] <Definition of Terms> In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Also, for a better understanding of the present disclosure, definitions and explanations of related terms are provided below. In the present disclosure, the term "pharmaceutically acceptable salt" refers to (i) a salt formed from an acidic functional group present in the compounds provided by the present disclosure and a suitable inorganic or organic cation (base), including but not limited to alkali metal salts such as sodium, potassium or lithium salts; alkaline earth metal salts such as calcium or magnesium salts; other metal salts such as aluminum, iron, zinc, copper, nickel or cobalt salts; inorganic alkali salts such as ammonium salts; organic alkali salts such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenylethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts, etc. Further, (ii) a salt formed from an acidic functional group present in the compounds provided by the present disclosure and a suitable inorganic or organic cation (base), including but not limited to alkali metal salts such as sodium, potassium or lithium salts; alkaline earth metal salts such as calcium or magnesium salts; other metal salts such as aluminum, iron, zinc, copper, nickel or cobalt salts; inorganic alkali salts such as ammonium salts; organic alkali salts such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N’-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzylphenylethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts, etc. Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of an alkaline compound with an appropriate acid that provides a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic compound with an appropriate base that provides a pharmaceutically acceptable cation.

[0019] In the present disclosure, the term "prodrug" refers to a derivative that can react by hydrolysis, oxidation, or other means under biological conditions (in vitro or in vivo) to provide a compound of the present disclosure. Prodrugs either provide the active compound only under biological conditions as a result of the reaction, or they are inactive or have lower activity in their unreacted form. Prodrugs can generally be prepared using well-known methods such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (edited by Manfred E. Wolff, 5th edition). Stereoisomers of the compounds described in the present disclosure should be understood to have the predominant configuration of the (R)-isomer or (S)-isomer, respectively, when specifically designated by chemical name as the (R)- or (S)-isomer. Any asymmetric carbon atom can be present in the (R)-, (S)-, or (R,S)-configuration, preferably in the (R)- or (S)-configuration. In the present disclosure, the terms "solvate" or "solvent compound" can be used interchangeably to refer to a compound that exists in combination with solvent molecules. This combination can include a stoichiometric amount of solvent, such as one molecule of water to form a monohydrate, or two molecules of water to form a dihydrate, or any amount of water; or it can include, for example, methanol or ethanol to form an "alcoholate", which can be stoichiometric or non-stoichiometric. As used herein, the term "solvate" refers to the solid form. That is, a compound that can be solvated, but a compound in a solvent solution is not a solvate as the term is used herein. In the present disclosure, the pharmaceutical excipient means an excipient and an additive used in the manufacture and formulation of a drug, and means a substance that is reasonably evaluated in terms of safety and is included in a pharmaceutical preparation in addition to the active ingredient. In addition to being used as an excipient and acting as a carrier to improve stability, it also has important functions such as solubilization, co-solubilization, and sustained and controlled release. Pharmaceutical excipients are important components that can affect the quality, safety, and efficacy of drugs. According to their sources, they can be classified into natural substances, semi-synthetic substances, and fully synthetic substances. According to their effects and uses, they can be classified into solvents, propellants, solubilizing agents, co-solvents, emulsifiers, coloring agents, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow promoters, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, encapsulating agents, moisturizing agents, absorbents, diluents, aggregating agents and peptizing agents, filter aids, release retardants, etc.

[0020] In the present disclosure, the pharmaceutical composition can be formulated into various suitable dosage forms according to the administration route, for example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powders, sprays, etc. Among these, the pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1,000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 mg to 500 mg, more preferably 0.5 mg to 350 mg, particularly preferably 1 mg to 250 mg of the antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate of the present disclosure. In the present disclosure, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, based on completely or partially preventing a disease or symptom; and / or therapeutic, based on partially or completely stabilizing or curing a disease and / or side effects caused by the disease. As used herein, "treatment" includes any treatment of a disease in a patient that (a) prevents the occurrence of a disease or symptom in a patient susceptible to the disease or symptom and not yet diagnosed; (b) suppresses the symptoms of a disease, i.e., stops its progression; or (c) alleviates the symptoms of a disease, i.e., delays the progression of the disease or symptom. In the present disclosure, the term "individual" includes human or non-human animals. Exemplary human individuals include human individuals (referred to as patients) suffering from a disease (e.g., a disease described herein) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates such as non-mammals (e.g., birds, amphibians, reptiles), as well as mammals such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.). In the present disclosure, the term "effective amount" refers to the amount of a compound that, when administered, alleviates some of one or more symptoms of the condition being treated. In the present disclosure, the term "antibody" should be interpreted in its broadest sense and includes full monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies as long as they have the desired biological activity. In the present disclosure, the terms "antibody" and "immunoglobulin" may be used interchangeably.

[0021] In the present disclosure, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies. That is, the antibodies constituting the group are identical except for a few natural variations that may exist. Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen. In contrast, polyclonal antibodies contain different antibodies against different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. In this specification, the modifier "monoclonal" should be understood to indicate that the antibody is characterized by being derived from a substantially homogeneous group of antibodies, and not to indicate that it is produced by a special method. In some embodiments of the present disclosure, the monoclonal antibody further includes, in particular, a chimeric antibody. That is, a part of the heavy chain and / or light chain is identical or homologous to a specific type, class, or subclass of the antibody, and the rest of the heavy chain and / or light chain is identical or homologous to another type, class, or subclass of the antibody as long as they have the desired biological activity (see, for example, US4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies consisting of variable region antigen-binding sequences derived from non-human primates (such as Old World monkeys, orangutans, etc.) and human constant region sequences. The monoclonal antibodies used in the present disclosure can be produced by a number of methods. For example, the monoclonal antibodies used in the present disclosure can be obtained by the hybridoma method using cells from a number of species (including cells derived from mice, hamsters, rats, and humans) (see, for example, Kohler et al., 1975, Nature, 256:495), or can be produced by recombinant DNA technology (see, for example, US4,816,567), or can be isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597). In the present disclosure, unless otherwise explicitly specified, the descriptors "each is independently selected from" and "each independently selected from" used throughout this specification can be used interchangeably, and should be interpreted in a broad sense, meaning that specific options represented by the same or different symbols in different groups do not affect each other, or specific options represented by the same or different symbols in the same group do not affect each other. In the present disclosure, "n1 is an integer selected from 1 to 5" means that n1 is selected from 1, 2, 3, 4, and 5. Other similar definitions can be understood by referring to the above.

[0022]

Chemical formula

[0023] In the present disclosure, L 3 is an amino acid residue or a peptide residue consisting of 2 to 10 amino acid residues, and the types of the 2 to 10 amino acids may be the same or different. For example, L 3 is a peptide residue consisting of 4 amino acid residues, and when the amino acids are selected from the group consisting of glycine and phenylalanine, L 3 may be a peptide residue such as glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), [Chemical formula] In each part of the present disclosure, the substituents of the compounds of the present disclosure are disclosed according to the type or range of the groups. It is specifically stated that all independent sub-combinations of these group types and ranges are included by the present disclosure. For example, the term "C1-C6 alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl. In the present disclosure, the term "C1-C6 alkyl group" refers to a straight-chain or branched-chain alkyl group containing 1 to 6 carbon atoms, for example, "C1-C3 alkyl group" or "C1-C4 alkyl group", including methyl group, ethyl group, etc. Specific examples include, but are not limited to, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, and hexyl group. In the present disclosure, the term "C1-C4 alkyl group" refers to a straight-chain or branched-chain alkyl group containing 1 to 4 carbon atoms, for example, "C1-C3 alkyl group", including methyl group, ethyl group, etc. Specific examples include, but are not limited to, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, and tert-butyl group.

[0024] Halogen refers to fluorine, chlorine, bromine or iodine. In the present disclosure, the term "halogenated C1-C6 alkyl group" means a group in which one or more hydrogen atoms in the above alkyl group (for example, C1-C6 alkyl group, C1-C4 alkyl group, C1-C3 alkyl group, etc.) are substituted with halogen (preferably fluorine, chlorine), and examples thereof include monofluoromethyl group, difluoroethyl group, trifluoromethyl group, etc. In the present disclosure, the term "deuterium-substituted C1-C6 alkyl group" means a group in which one or more hydrogen atoms in any of the above alkyl groups (for example, C1-C6 alkyl group, C1-C4 alkyl group, C1-C3 alkyl group, etc.) are substituted with deuterium atoms, and examples thereof include monodeuteromethyl, dideuteromethyl, trideuteromethyl, etc. Those skilled in the art can understand that in the antibody-drug conjugate disclosed in the present disclosure, the antibody is linked to the linker-payload via -S-, and -S- is not an additional external sulfhydryl group, but a sulfhydryl group generated by the antibody itself after reduction and opening of the disulfide bond. In the present disclosure, the drug-to-antibody ratio (DAR) refers to the number of drug molecules bound to an antibody (for example, a in Formula I). The number of drug molecules contained in the antibody-drug conjugate described herein can be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules conjugated per antibody. The term "a is any number between 1 and 10" means that a can be any integer selected from 1 to 10 (including the endpoints 1 and 10), or any decimal selected from between 1 and 10 such as 3.9 or 4.0. At the same time, even when the same preparation method is used, the DAR values of the antibody-drug conjugates obtained from different preparation batches may not necessarily be exactly the same. For example, it can be understood by those skilled in the art that they can vary up and down within 0.5. In the present disclosure, the term "about" is understood to be within + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, + / - 0.5%, + / - 0.4%, + / - 0.3%, + / - 0.2%, + / - 0.1% of the stated value. All values provided herein are modified by the term "about" unless it is clear from the context.

[0025] In the present disclosure, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the necessary properties of a protein / polypeptide containing an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the substitution of an amino acid residue with another amino acid residue having a similar side chain, for example, substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having chemical properties such as similar size, shape, charge, ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, for example, Brummell et al. Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burksra, Proc. Natl Acad. Sci. USA 94:412-417 (1997)).

Advantages of the Invention

[0026] 1. The ADCs of the present disclosure exhibit excellent inhibitory activity against multiple tumor cell lines having different target expression levels. 2. The ADC of the present disclosure exhibits a potent in vivo growth inhibitory effect in a subcutaneous xenograft tumor model of nude mice having human-derived tumor cells.

Brief Description of the Drawings

[0027] The drawings described herein are used to provide a further understanding of the present disclosure, form a part of this application, and the exemplary embodiments of the present disclosure and their descriptions are used to explain the present disclosure and do not constitute an undue limitation of the present disclosure. The drawings are as follows.

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0028] Hereinafter, the present disclosure will be further described with specific examples, but the present disclosure is not limited thereto. Various modifications or improvements may be made by those skilled in the art in accordance with the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure. Reagents or equipment used without the instructions of the manufacturer are commercially available conventional products. In the following embodiments, some abbreviations of drugs and their structural formulas correspond as follows.

Chemical

[0029] In the following examples, the antibodies used are known antibodies, and the sequences of the antibodies are as shown below. The CDRs (underlined) are defined as follows using the Kabat method. Antibody P7-C05-H4L3 sequence (see WO2021244590A1): <Heavy chain> Formula 1

[0030] JPEG2025524970000021.jpg50164 Here, from left to right, the non-shaded grey part is the heavy chain variable region sequence (SEQ ID NO: 8), and the shaded grey part is the heavy chain constant region sequence. In the heavy chain variable region sequence, the underlined parts are the heavy chain CDR1 sequence (SEQ ID NO: 5), the heavy chain CDR2 sequence (SEQ ID NO: 6), and the heavy chain CDR3 sequence (SEQ ID NO: 7) in order from the left. <Light chain> Formula 2

[0031] JPEG2025524970000022.jpg28164 Here, from left to right, the non-shaded grey part is the light chain variable region sequence (SEQ ID NO: 12), and the shaded grey part is the light chain constant region sequence. In the light chain variable region sequence, the underlined parts are the light chain CDR1 sequence (SEQ ID NO: 9), the light chain CDR2 sequence (SEQ ID NO: 10), and the light chain CDR3 sequence (SEQ ID NO: 11) in order from the left.

[0032] The sequence of control ADC2 (infliximab) (see Reference Example 1 of CN 104755494 A, i.e., M30-H1-L4) is as follows. <Heavy chain> Formula 3

[0033] JPEG2025524970000023.jpg54164 Here, from left to right, the non-shaded grey part is the heavy chain variable region sequence (SEQ ID NO: 16), and the shaded grey part is the heavy chain constant region sequence. In the heavy chain variable region sequence, the underlined parts are the heavy chain CDR1 sequence (SEQ ID NO: 13), the heavy chain CDR2 sequence (SEQ ID NO: 14), and the heavy chain CDR3 sequence (SEQ ID NO: 15) in order from the left. <Light chain> Formula 4

[0034] JPEG2025524970000024.jpg27164 Here, from left to right, the non - gray - shaded part is the light - chain variable - region sequence (SEQ ID NO: 20), and the gray - shaded part is the light - chain constant - region sequence. In the light - chain variable - region sequence, the underlined parts are the light - chain CDR1 sequence (SEQ ID NO: 17), the light - chain CDR2 sequence (SEQ ID NO: 18), and the light - chain CDR3 sequence (SEQ ID NO: 19) in order from left.

[0035] This disclosure is further illustrated and exemplified by reference to the following specific examples. Unless otherwise specified, the reaction materials are commercially available and obtainable.

Example

[0036] Preparation of Linker - Payload 1.1 Preparation of Compound II - 12 - a

Chemical formula

Chemical formula

[0037] Step 1 Compound 1a (50.00 g, 135.90 mmol) and benzyl glycolate (45.00 g, 271.70 mmol) were successively added to a three-necked flask (1 L). Subsequently, the atmosphere was replaced with N2, and anhydrous tetrahydrofuran (500 mL) was added. Then, the flask was placed in an ice bath, and anhydrous p-toluenesulfonic acid (2.30 g, 13.40 mmol) was added, and the resulting reaction mixture was stirred under the ice bath for 2 hours. Water (1 L) and ethyl acetate (1 L) were added to the reaction mixture, and the layers were separated. The organic phase was washed with an aqueous NaHCO3 solution. Aqueous sodium bicarbonate, water, and aqueous sodium chloride were successively added. The organic phase was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under vacuum, and the residue was purified by flash column chromatography on silica gel (methanol:dichloromethane = 0 - 100%) to obtain Compound 1b (27.00 g) in a yield of 42%. MS-ESI "M+Na"+: calculated value 497, measured value: 497. Step 2 Compound 1b (17.00 g, 35.86 mmol) was dissolved in N,N-dimethylacetamide (170 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (2.78 g, 18.29 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour under a N2 atmosphere. Pyridinium p-toluenesulfonate (4.59 g, 18.29 mmol), 1-hydroxybenzotriazole (4.59 g, 34.00 mmol), 1c (16.66 g, 33.18 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.37 g, 33.18 mmol) were added to the above reaction mixture. After the addition was complete, the resulting mixture was stirred at room temperature for an additional 2.5 hours. 2-Methyltetrahydrofuran (175 mL) and saturated aqueous sodium chloride solution (260 mL) were added to the saturated reaction mixture aqueous solution, and the organic phase was extracted and separated. The aqueous phase was extracted with 2-methyltetrahydrofuran (100 mL × 2). The obtained organic phases were combined. The combined organic phases were washed successively with 10% citric acid (90 mL), aqueous sodium hydrogen carbonate solution three times (175 mL × 3), and saturated brine once (100 mL), dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated to about 50 mL under vacuum, and isopropanol (120 mL) was added. The resulting solution was reconcentrated to about 50 mL under vacuum, and then isopropanol (300 mL) was added. The resulting solution was stirred at 60 °C for 1 hour, then cooled to 0 - 5 °C, stirred at 0 - 5 °C for 2 hours, and filtered. The obtained solid was washed with isopropanol (5 °C) to obtain Compound 1d (19.00 g) in a yield of 78%. Step 3 Compound 1d (11.50 g, 15.65 mmol) was dissolved in a mixed solvent of tetrahydrofuran (230 mL) and H2O (115 mL), and 1,8-diazabicyclo[5.4.0]undec-7-ene (10.70 g, 70.39 mmol) was added to the resulting solution. The mixture was stirred at room temperature for 4 hours under a N2 atmosphere. The reaction mixture was extracted 5 times (100 mL × 5) with tert-butyl methyl ether. The aqueous phase was concentrated to obtain a yellow oily residue, which was subsequently dissolved in isopropanol (70 mL). The resulting solution was slowly added to tert-butyl methyl ether (700 mL). A white solid precipitated, was filtered, and collected. The obtained white solid was dissolved in ethanol and concentrated under vacuum to obtain white foamy solid 1e (9.50 g) in a yield of 93%. Step 4 Compound 1e (1.00 g, 1.53 mmol) was dissolved in a mixed solvent of acetonitrile (10 mL) and H2O (20 mL), and compound 1f (0.54 g, 1.75 mmol) was added to the resulting solution. The mixture was stirred at room temperature for 5 hours under a N2 atmosphere. Water (20 mL) was added to the mixture, and then the pH was adjusted to 3 - 4 by adding 0.5 N HCl (aqueous solution). The resulting mixture was extracted 4 times with a mixed solvent of isopropanol / dichloromethane (V:V = 4:1, 50 mL). The obtained organic phases were combined. The combined organic phases were dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated under vacuum to obtain a yellow oily residue, which was subsequently dissolved in a mixed solvent of dichloromethane / methanol (v:v = 4:1, 5 mL). The resulting solution was slowly added to tert-butyl methyl ether (100 mL). A white solid precipitated, was filtered, and collected to obtain compound 1g (850 mg) in a 90% yield. MS-ESI "M+Na"+: calculated value 639, measured value: 639.

[0038] Step 5 Compound 1h (12.20 g, 46.39 mmol) was added to a three-necked flask (500 mL). The reaction mixture was cooled to room temperature and the atmosphere was replaced with N2 (gas). Subsequently, the flask was placed in an ice bath. N,N-Dimethylformamide (120 mL) was added, followed by the sequential addition of imidazole (15.77 g, 232.00 mmol) and chlorotriethylsilane (27.97 g, 185.56 mmol). The resulting mixture was stirred for 10 minutes, and then 4-dimethylaminopyridine (5.66 g, 46.39 mmol) was added. The resulting mixture was stirred at 0 °C for 3 hours. The reaction mixture was diluted with tert-butyl methyl ether (1.20 L), followed by the addition of water (1.2 L), and the layers were separated. The aqueous phase was extracted with tert-butyl methyl ether (1.2 L). The obtained organic phases were combined. The combined organic phases were washed once with water (1.20 L), dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (ethyl acetate: petroleum ether = 0-100%) to obtain crude 1i (16.00 g). Step 6 To a three-necked flask (1 L), crude 1i (16.00 g, 32.59 mmol) and Lawesson's reagent (13.18 g, 32.59 mmol) were sequentially added, followed by the addition of anhydrous toluene (600 mL). Subsequently, the atmosphere was replaced with N2, and the reaction mixture was refluxed at 125 °C for 5 hours. The reaction mixture was concentrated, and the residue was purified by flash column chromatography on silica gel (ethyl acetate: petroleum ether = 0-100%) to obtain crude 1j (16.00 g). Step 7 The crude 1j (16 g of crude) was dissolved in anhydrous tetrahydrofuran (640 mL), and triethylamine trihydrofluoride (11.93 g, 74.00 mmol) was added dropwise to the resulting solution under an ice bath. After the addition was complete, the resulting mixture was slowly warmed to room temperature and stirred overnight. Subsequently, the reaction mixture was diluted with ethyl acetate (1 L), and then the layers were separated. The organic phase was washed with saturated brine (500 mL × 2), dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (ethyl acetate: petroleum ether = 0 - 100%) to obtain compound 1k (6.38 g) in a three-step yield of 49%. Step 8 To a three-necked flask (500 mL), compound 1l (3.00 g, 12.00 mmol), 1k (4.33 g, 15.52 mmol), pyridinium p-toluenesulfonate (3.90 g, 15.50 mmol), and o-cresol (15 mL) were added, followed by anhydrous toluene (300 mL). Subsequently, the atmosphere was replaced with N2, and the reaction mixture was stirred at 120 °C under an oil bath for 32 h. The reaction mixture was cooled to room temperature, and the insoluble matter was collected by filtration. The obtained crude product was triturated with tert-butyl methyl ether (60 mL) for 1 h. Subsequently, the mixture was filtered and dried to obtain compound 1m (3.0 g) in a yield of 51%.

[0039] Step 9 Compound 1m (2.30 g, 4.67 mmol) was added to a single-necked flask (500 mL), followed by 6N HCl (aqueous solution) (230 mL). The resulting mixture was refluxed at 110 °C for 12 h. The reaction mixture was hot-filtered to remove the insoluble substances. The filtrate was concentrated under vacuum to obtain the crude product 1n (a pair of diastereomers). The crude product 1n was purified by preparative HPLC to obtain compound 1n-P1 (412 mg, the isomer with lower polarity in TLC) and 1n-P2 (495 mg, the isomer with higher polarity in TLC) in a yield of 34%. Step 10 Synthesis of Compound 1n-P2 Into a one-necked flask (10 mL), Compound 1n-P1 (50 mg, 0.09 mmol), 1g (76 mg, 0.12 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (31 mg, 0.16 mmol) and N,N-dimethylformamide (2 mL) were successively added, and then 2,4,6-trimethylpyridine (60 mg, 0.49 mmol) was added dropwise. After the addition was completed, the resulting mixture was stirred at room temperature for 3 hours. The reaction solution was added dropwise to tert-butyl methyl ether (60 mL). A solid precipitated, was filtered and recovered. The obtained solid was dissolved in a mixed solvent of dichloromethane / methanol (V:V = 6:1, 30 mL). The obtained solution was concentrated, and the resulting crude product was purified by preparative HPLC to obtain Compound II-12-a (33 mg) in a yield of 35%. MS-ESI "M+H": Calculated value 1050, Measured value: 1050. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (t, J = 6.4 Hz, 1H), 8.60 (d, J = 8.8 Hz, 1H), 8.28 (t, J = 5.6 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.06 (t, J = 6.0 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.78 (s, 1H), 7.28 - 7.14 (m, 5H), 6.97 (s, 1H), 5.46 (d, J = 2.8 Hz, 1H), 5.30 (d, J = 19.6 Hz, 1H), 4.67 (d, J = 6.4 Hz, 1H), 4.51 - 4.41 (m, 1H), 4.18 - 4.04 (m, 2H), 3.77 - 3.73 (m, 1H), 3.73 - 3.68 (m, 2H), 3.68 - 3.62 (m, 2H), 3.62 - 3.58 (m, 1H), 3.58 - 3.54 (m, 1H), 3.38 - 3.31 (m, 2H), 3.30 - 3.20 (m, 1H), 3.20 - 3.08 (m, 1H), 3.01 (dd, J = 14.0, 4.8 Hz, 1H), 2.80 - 2.71 (m, 1H), 2.37 (s, 3H), 2.26 - 2.15 (m, 2H), 2.08 (t, J = 7.6 Hz, 2H), 1.94 - 1.82 (m, 2H), 1.54 - 1.37 (m, 4H), 1.25 - 1.10 (m, 2H), 0.85 (t, J = 7.6 Hz, 3H).

[0040] 1.2 Preparation of Compound II-1-a

Chem.

Chem.

[0041] Step 1 Compound 2a (1.00 g, 7.52 mmol) was dissolved in N,N-dimethylformamide (10 mL), and maleic anhydride (0.74 g, 7.52 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 16 h under a N2 atmosphere. The resulting reaction solution was recorded as solution A and stored. N-Hydroxysuccinimide (3.46 g, 30.08 mmol) and N,N-dimethylformamide (10 mL) were added to a three-necked flask (50 mL), the mixture was cooled to 0 °C, and then trifluoroacetic anhydride (6.31 g, 30.08 mmol) was added dropwise. After the addition was complete, the reaction mixture was stirred for 0.5 h, and then 2,4,6-trimethylpyridine (5.46 g, 45.10 mmol) was added. After the addition was complete, the reaction mixture was stirred for an additional 0.5 h. Then, the reaction solution was added dropwise to the above solution A. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was adjusted to pH 2 - 3 by adding 1 N HCl (aqueous solution) and extracted with dichloromethane (50 mL). The organic phase was washed three times with water, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (ethyl acetate:petroleum ether = 0 - 100%) to give compound 2b (1.20 g) in 51% yield. Step 2 Compound 2b (0.40 g, 1.29 mmol) was dissolved in a mixed solvent of acetonitrile (8 mL) and water (16 mL), and compound 1e (0.54 g, 0.80 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 16 h under a N2 atmosphere. The reaction mixture was concentrated under vacuum, and the residue was purified by preparative HPLC to give compound 2c (0.30 g) in 61% yield. Step 3 Compound 1 n-P1 (50 mg, 0.09 mmol), 2c (76 mg, 0.12 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (31 mg, 0.16 mmol), and N,N-dimethylformamide (2 mL) were sequentially added to a one-neck flask (10 mL), and 2,4,6-trimethylpyridine (60 mg, 0.49 mmol) was added dropwise. After the addition was completed, the resulting mixture was stirred at room temperature for 3 hours. The reaction solution was added dropwise to tert-butyl methyl ether (60 mL). A solid precipitated, was filtered, and collected. The obtained solid was dissolved in a mixed solvent of dichloromethane / methanol (V:V = 6:1, 30 mL). The obtained solution was concentrated, and the resulting crude product was purified by preparative HPLC to obtain Compound II-1-a (16 mg) in a yield of 17%. MS-ESI "M+H"+: calculated value 1052, measured value: 1052. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (t, J = 6.8 Hz, 1H), 8.61 (d, J = 9.2 Hz, 1H), 8.28 (t, J = 4.4 Hz, 1H), 8.14 - 8.06 (m, 2H), 8.06 (t, J = 5.6 Hz, 1H), 7.82 (d, J = 11.2 Hz, 1H), 7.79 (s, 1H), 7.28 - 7.13 (m, 5H), 6.99 (s, 2H), 5.92 (d, J = 16.8 Hz, 1H), 5.70 - 560 (m, 1H), 5.53 (d, J = 8.0 Hz, 1H), 5.48 (d, J = 4.8 Hz, 1H), 5.37 (d, J = 19.6 Hz, 1H), 4.68 (d, J = 6.4 Hz, 1H), 4.51 - 4.41 (m, 1H), 4.17 - 4.04 (m, 2H), 3.72 - 3.68 (m, 2H), 3.68 - 3.63 (m, 2H), 3.62 - 3.58 (m, 1H), 3.58 - 3.54 (m, 3H), 3.54 - 3.39 (m, 3H), 3.48 - 3.43 (m, 2H), 3.19 - 3.08 (m, 1H), 3.06 - 2.97 (m, 1H), 2.80 - 2.70 (m, 1H), 2.70 - 2.65 (m, 1H), 2.39 (s, 3H), 2.35 - 2.28 (m, 3H), 2.27 - 2.18 (m, 2H), 1.94 - 1.82 (m, 2H), 0.85 (t, J = 7.2 Hz, 3H).

[0042] 1.3 Preparation of Compound II-3-a

Chem.

Chem.

[0043] Project 1 Compound 3a (10.00 g, 95.11 mmol) was dissolved in N,N-dimethylformamide (100 mL), and maleic anhydride (9.32 g, 95.11 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 16 h under a N2 atmosphere. Water (250 mL) was added to the reaction mixture, and the aqueous phase was extracted with dichloromethane / isopropanol (v:v = 4:1, 200 mL × 3). The combined organic phases were concentrated under vacuum to obtain crude 3b (12.00 g). Project 2 Crude 3b (4.00 g, 31.70 mmol) was dissolved in N,N-dimethylformamide (60 mL), and bis(4-nitrophenyl) carbonate (16.40 g, 53.95 mmol) was added to the resulting solution. The mixture was heated to 30 °C, and subsequently N,N-diisopropylethylamine (4.18 g, 32.40 mmol) was added. The reaction was then stirred for 2 h. The reaction solution was cooled to room temperature, water (600 mL) was added, and the aqueous phase was extracted with dichloromethane (300 mL × 2). The obtained organic phases were combined. The combined organic phases were dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (ethyl acetate:petroleum ether = 0 - 100%) to obtain compound 3c (2.40 g) in a two-step yield of 13%. Project 3 Compound 3c (400 mg, 1.14 mmol) was dissolved in a mixed solution of acetonitrile (8 mL) and water (16 mL), compound 1e (760 mg, 1.13 mmol) was added, and the mixture was stirred at room temperature for 16 h under a N2 atmosphere. The reaction mixture was directly purified by preparative HPLC to obtain compound 3d (109 mg) in a yield of 15%. Project 4 Compound 1 n-P1 (69 mg, 0.12 mmol), 3d (110 mg, 0.17 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (44 mg, 0.23 mmol) and N,N-dimethylformamide (2.5 mL) were successively added to a one-necked flask (10 mL), and then 2,4,6-trimethylpyridine (84 mg, 0.69 mmol) was added dropwise to the resulting mixture. The resulting mixture was stirred at room temperature for 3 hours. The resulting reaction solution was added dropwise to tert-butyl methyl ether (75 mL). A solid precipitated, was filtered and collected. The obtained solid was dissolved in a mixed solvent of dichloromethane / methanol (V:V = 6:1, 30 mL). The obtained solution was concentrated in vacuo, and the residue was purified by preparative HPLC to obtain Compound II-3-a (45 mg) in a yield of 35%. MS-ESI "M+H"+: calculated value 1068.4, measured value: 1068.9. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (t, J = 6.8 Hz, 1H), 8.62 (d, J = 9.2 Hz, 1H), 8.31 (t, J = 5.6 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.78 (s, 1H), 5.90 (d, J = 16.4 Hz, 1H), 7.27 - 7.13 (m, 5H), 6.99 (s, 1H), 5.46 (d, J = 4.4 Hz, 1H), 5.29 (d, J = 19.6 Hz, 1H), 4.67 (d, J = 6.4 Hz, 1H), 4.51 - 4.41 (m, 1H), 4.17 - 4.04 (m, 2H), 4.02 - 3.94 (m, 2H), 3.79 - 3.67 (m, 3H), 3.35 - 3.20 (m, 1H), 3.20 - 3.07 (m, 1H), 3.00 (dd, J = 14.0, 4.4 Hz, 1H), 2.78 - 2.65 (m, 1H), 2.37 (s, 3H), 2.25 - 2.15 (m, 2H), 1.92 - 1.82 (m, 2H), 0.85 (t, J = 7.2 Hz, 3H).

[0044] 1.4 Preparation of Compound II-2-a

Chemical Structure

Chem.

[0045] Project 1 Compound 4a (2.00 g, 7.52 mmol) and β-alanine (0.67 g, 7.52 mmol) were dissolved in acetonitrile (20 mL). Then the mixture was cooled to 0 °C, and subsequently N,N-diisopropylethylamine (1.94 g, 15.03 mmol) was added. After the addition was complete, the reaction mixture was returned to room temperature and stirred for 16 h. Water (100 mL) was added to the reaction mixture, and then the reaction mixture was extracted with dichloromethane / isopropanol (v:v = 4:1, 250 mL × 10). The combined organic phases were concentrated, and the residue was purified by flash column chromatography on silica gel (methanol:dichloromethane = 0 - 100%) to obtain Compound 4b (2.00 g). Project 2 Compound 4b (1.80 g, 7.50 mmol) was dissolved in N,N-diisopropylethylamine (8 mL), and dicyclohexylcarbodiimide (1.85 g, 9.00 mmol) was added to the resulting solution. After the addition was complete, the reaction mixture was stirred for 30 min, and subsequently N-hydroxysuccinimide (0.95 g, 8.25 mmol) was added. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was filtered, and the filtrate was directly purified by preparative HPLC to obtain Compound 4c (530 mg) in a yield of 21%. Project 3 Compound 4c (0.50 g, 1.48 mmol) was dissolved in a mixed solvent of acetonitrile (8 mL) and water (16 mL), and 1e (0.98 g, 1.50 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 16 h under a N2 atmosphere. The reaction mixture was directly purified by preparative HPLC to obtain Compound 4d (42 mg) in a yield of 4%. Project 4 Compound 1 n-P1 (26 mg, 0.046 mmol), 4d (42 mg, 0.065 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16 mg, 0.083 mmol) and N,N-dimethylformamide (1 mL) were sequentially added to a one-necked flask (10 mL), and then 2,4,6-trimethylpyridine (32 mg, 0.26 mmol) was added dropwise to the mixture. The resulting mixture was reacted at room temperature for 3 hours. The reaction solution was added dropwise to tert-butyl methyl ether (30 mL). A solid precipitated, was filtered and recovered. The obtained solid was dissolved in a mixed solvent of dichloromethane / methanol (V:V = 6:1, 30 mL). The obtained solution was concentrated in vacuo, and the residue was purified by preparative HPLC to obtain Compound II-2-a (13 mg) in a yield of 26%. MS-ESI "M+H": Calculated value 1079.4, Measured value: 1079.9. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (t, J = 6.8 Hz, 1H), 8.63 (d, J = 8.8 Hz, 1H), 8.31 (t, J = 6.0 Hz, 1H), 8.18 (t, J = 7.2 Hz, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.03 (t, J = 5.6 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.82 (d, J = 10.8 Hz, 1H), 7.79 (s, 1H), 7.28 - 7.13 (m, 5H), 6.98 (s, 2H), 6.70 (s, 1H), 5.90 (d, J = 16.8 Hz, 1H), 5.70 - 560 (m, 1H), 5.56 - 5.45 (m, 2H), 5.36 (d, J = 19.6 Hz, 1H), 4.68 (d, J = 6.8 Hz, 1H), 4.53 - 4.42 (m, 1H), 4.18 - 4.04 (m, 2H), 3.78 - 3.64 (m, 5H), 3.62 - 3.52 (m, 4H), 3.22 - 3.14 (m, 1H), 3.06 - 2.97 (m, 1H), 2.80 - 2.70 (m, 1H), 2.68 - 2.65 (m, 1H), 2.39 (s, 3H), 2.34 - 2.16 (m, 6H), 1.92 - 1.82 (m, 2H), 0.85 (t, J = 7.2 Hz, 3H).

[0046] 1.5 Preparation of Compound II-12-b

Chem.

Chem.

[0047] Step 1 Under N2 protection, at 0 °C, 1n (24.00 mg, 0.053 mmol) and 1g (32.78 mg, 0.053 mmol) were dissolved in N,N-dimethylformamide (1.5 mL), and then a solution of N,N-diisopropylethylamine (17.10 mg, 0.0133 mmol) in N,N-dimethylformamide (0.4 mL) and a solution of 2-(7-azabenzotriazol-1-yl)-N,N,N‘,N’-tetramethyluronium hexafluorophosphate (24.20 mg, 0.064 mmol) in N,N-dimethylformamide (0.6 mL) were successively added dropwise. After the addition was completed, the resulting reaction mixture was maintained at 0 °C and reacted for 1 hour. The reaction mixture was purified to obtain Compound II-12-a (16 mg) and II-12-b (19 mg) in an overall yield of 64%. II-12-b: MS-ESI "M+H"+: Calculated value 1050.4, Measured value: 1050.3. 1H NMR (400 MHz, DMSO-d6) δ 8.71 - 8.61 (m, 2H), 8.31 (t, J = 4.8 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.07 (t, J = 6.0 Hz, 1H), 8.00 (t, J = 5.2 Hz, 1H), 7.82 (dd, J = 10.8, 2.0 Hz, 1H), 7.80 (s, 1H), 7.28 - 7.13 (m, 6H), 6.99 (s, 2H), 6.70 (br s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.70 - 561 (m, 1H), 5.52 (d, J = 17.2 Hz, 2H), 5.37 (d, J = 19.6 Hz, 1H), 4.68 (d, J = 6.8 Hz, 2H), 4.51 - 4.41 (m, 1H), 4.18 - 4.04 (m, 2H), 3.77 - 3.50 (m, 7H), 3.32 - 3.20 (m, 1H), 3.20 - 3.08 (m, 1H), 3.07 - 2.96 (m, 1H), 2.82 - 2.71 (m, 1H), 2.39 (s, 3H), 2.26 - 2.15 (m, 2H), 2.09 (t, J = 7.6 Hz, 2H), 1.96 - 1.82 (m, 2H), 1.52 - 1.39 (m, 4H), 1.25 - 1.10 (m, 2H), 0.85 (t, J = 7.6 Hz, 3H).

[0048] 1.6 Preparation of Compounds X and Y

Chemical Structure

[0049] Step 1 To a one-necked flask (25 mL), 1n (20 mg, 0.04 mmol) and a solution of glycolic acid in N,N-dimethylformamide (2.00 mL, 0.032 mmol, 1.2 mg / mL) were added. The mixture was cooled to 0 °C, and N,N-diisopropylethylamine (11 mg, 0.08 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N‘,N’-tetramethyluronium hexafluorophosphate (16 mg, 0.04 mmol) were added in that order. The reaction mixture was reacted for 0.5 h. A separate batch of a solution of glycolic acid in N,N-dimethylformamide (1.2 mg / mL, 0.20 mL, 0.003 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N‘,N’-tetramethyluronium hexafluorophosphate (1 mg) were added, and then the reaction mixture was reacted for an additional 1 h. The reaction mixture was diluted with ethyl acetate (80 mL). The organic phase was washed successively with 0.5 N HCl(aq) (5 mL×1), saturated aqueous sodium hydrogen carbonate solution (10 mL×2), and saturated brine (10 mL×5). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under vacuum, and the residue was purified by preparative TLC (methanol:dichloromethane) to obtain compound X (2 mg) and Y (5 mg) in an overall yield of 34%. X: MS-ESI "M+H"+: calculated value 510, measured value: 510. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 10.8 Hz, 1H), 7.80 (s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.17.60 (m, 1H), 5.53 (d, J = 20.4 Hz, 1H), 5.52 (d, J = 16.4 Hz, 1H), 5.35 (d, J = 20.0 Hz, 1H), 4.15 - 3.98 (m, 2H), 3.30 - 3.22 (m, 1H), 3.19 - 3.09 (m, 1H), 2.40 (s, 3H), 2.26 - 2.14 (m, 2H), 1.96 - 1.84 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H). Y: MS-ESI "M+H"+: Calculated value 510, measured value: 510. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 9.2 Hz, 1H), 7.81 (d, J = 11.2 Hz, 1H), 7.80 (s, 1H), 6.69 (s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.69 - 5.62 (m, 1H), 5.62 - 5.56 (m, 1H), 5.55 - 5.47 (m, 2H), 5.33 (d, J = 20.0 Hz, 1H), 4.16 - 3.98 (m, 2H), 3.30 - 3.20 (m, 1H), 3.16 - 3.07 (m, 1H), 2.39 (s, 3H), 2.26 - 2.15 (m, 2H), 1.95 - 1.85 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

Example

[0050] Preparation and testing of antibody-drug conjugates 1. General preparation method The antibody was dialyzed in 50 mM PB buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution were successively added, and additional 50 mM PB buffer was added to make the final concentration of the antibody in the reaction system 5 - 10 mg / mL, the molar ratio of TCEP to antibody (2.2 - 8.0):1.0, and the final concentration of DTPA 0.5 - 1.5 mM. After the reaction system was thoroughly mixed, the reduction reaction temperature was set at 25 ± 2 °C, and it was placed in a thermostatic mixer with a rotation speed of 400 rpm for a reduction reaction for 1.0 - 2.5 hours. After the completion of the reduction reaction, an appropriate amount of 5 mM linker-payload stock solution was successively added to each reaction system under ice bath. The molar ratio of linker-payload to antibody was 4.5 - 12.0. If the purity of the conjugate product was low, supplementary addition of DMSO and / or arginine hydrochloride might be considered. After the reaction system was thoroughly mixed, it was placed in a thermostat mixer with a conjugation reaction temperature of 25 ± 2 °C and a rotation speed of 400 rpm for a conjugation reaction for 0.5 - 1.5 hours. After the completion of the conjugation reaction, the ADC sample was dialyzed into a dialysis solution (10 mM or 20 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifugal tube to obtain an ADC stock solution, which was aliquoted and a certain amount was stored at -80 °C.

[0051] Preparation of 1.1M H-ADC1

Chemical formula

[0052] 1.2 Preparation of MH-ADC2

Chemical formula

[0053] 1.2.1 Preparation of MH-ADC2-1 The antibody P7-C05-H4L3 was dialyzed in 50 mM PB (pH 7.4 ± 0.2) buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and appropriate amounts of 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution were successively added, and additional 50 mM PB (pH 7.4 ± 0.2) buffer was added to make the final concentration of the antibody in the reaction system 10 mg / mL, the molar ratio of TCEP to antibody 2.6:1.0, and the final concentration of DTPA 1 mM. After the reaction system was thoroughly mixed, the reduction reaction temperature was set at 25 ± 2 °C, and it was placed in a thermostatic mixer with a rotation speed of 400 rpm for a 2-hour reduction reaction. Linker-payload II-1-a was prepared as a 5 mM stock solution in DMSO. After completion of the reduction reaction, an appropriate amount of the 5 mM stock solution of linker-payload II-1-a was added to the reaction system under an ice bath so that the molar ratio of linker-payload to antibody was 5.5. The reaction system was mixed, and then the conjugation reaction temperature was set at 25 ± 2 °C and the rotation speed at 400 rpm for a 1-hour conjugation reaction. After completion of the conjugation reaction, the obtained ADC sample was dialyzed into a dialysis solution (10 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifugal tube to obtain an MH-ADC2-1 stock solution, which was aliquoted and a certain amount was stored at -80 °C.

[0054] 1.2.2 Preparation of MH-ADC2-2 The antibody P7-C05-H4L3 was dialyzed in 50 mM PB (pH 7.4 ± 0.2) buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and an appropriate amount of 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution were continuously added, and additional 50 mM PB (pH 7.4 ± 0.2) buffer was added to make the final concentration of the antibody in the reaction system 5 mg / mL, the molar ratio of TCEP to the antibody 2.2:1.0, and the final concentration of DTPA 1 mM. After thoroughly mixing the reaction system, it was placed in a thermostatic mixer with a reduction reaction temperature of 25 ± 2 °C and a rotation speed of 400 rpm, and a reduction reaction was carried out for 1 hour. The linker-payload II-1-a was prepared as a 5 mM stock solution in DMSO. After completion of the reduction reaction, an appropriate amount of the 5 mM stock solution of the linker-payload II-1-a was added to the reaction system under an ice bath so that the molar ratio of the linker-payload to the antibody was 5.0. After thoroughly mixing the reaction system, it was placed in a thermostat mixer at a conjugation reaction temperature of 25 ± 2 °C and a rotation speed of 400 rpm and subjected to a conjugation reaction for 1 hour. After completion of the conjugation reaction, the obtained ADC sample was dialyzed into a dialysis solution (10 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifugal tube to obtain an MH-ADC2-2 stock solution, which was divided and stored at -80 °C.

[0055] 1.2.3 Preparation of MH-ADC2-3 The antibody P7-C05-H4L3 was dialyzed in 50 mM PB (pH 7.7 ± 0.2) buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and an appropriate amount of 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution were continuously added, and additional 50 mM PB (pH 7.7 ± 0.2) buffer was added to make the final concentration of the antibody in the reaction system 10 mg / mL, the molar ratio of TCEP to antibody 3.5:1.0, and the final concentration of DTPA 1 mM. After the reaction system was thoroughly mixed, it was placed in a thermostatic mixer with a reduction reaction temperature of 25 ± 2 °C and a rotation speed of 400 rpm, and a reduction reaction was carried out for 2 hours. The linker-payload II-1-a was prepared as a 5 mM stock solution in DMSO. After the completion of the reduction reaction, an appropriate amount of the 5 mM stock solution of the linker-payload II-1-a was added to the reaction system under an ice bath so that the molar ratio of the linker-payload to the antibody was 7.0. After the reaction system was thoroughly mixed, it was placed in a thermostat mixer with a conjugation reaction temperature of 25 ± 2 °C and a rotation speed of 400 rpm and subjected to a conjugation reaction for 1 hour. After the completion of the conjugation reaction, the obtained ADC sample was dialyzed into a dialysis solution (10 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifugal tube to obtain an MH-ADC2-3 stock solution, which was aliquoted and a certain amount was stored at -80 °C.

[0056] 1.3 Preparation of control ADC1

Chemical formula

[0057] 1.4 Preparation of control ADC2

Chemical formula

[0058] 2. Detection of the drug-to-antibody ratio of the antibody-drug conjugate The average drug-to-antibody ratio (DAR) of the ADC was detected by high-speed liquid hydrophobic interaction chromatography (HIC-HPLC). The ADC sample was equilibrated to room temperature and thoroughly mixed. Then, ultrapure water was used as the diluent to dilute the sample to a target concentration of 5 mg / mL according to the protein concentration, and it was mixed for 20 seconds with a vortex mixer. The diluted sample solution was pipetted into the injection tube, and the sample number was marked on the injection bottle. After equilibration, the sample was detected using a high-speed liquid chromatograph (Agilent, 1260 Bio) and a hydrophobic column (TOSOH, TSKgel Butyl-NPR column (2.5) 4.6 mm × 35 mm). The main parameters of the acquisition method are shown in Tables 1 and 2. Mobile phase A was formulated as 20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0, and mobile phase B was formulated as 20 mM sodium phosphate, 25% isopropanol (v / v), pH 7.0. The HPLC spectrum was integrated. Then, using the integration results, the percentages of ADCs with 2, 4, 6, and 8 drugs bound and unbound ADCs (DAR0%, DAR2%, DAR4%, DAR6%, DAR8% respectively) were determined, and the average drug-to-antibody ratio was calculated using the following formula, and the results are shown in Table 3. DAR value = (0 × DAR0% + 2 × DAR2% + 4 × DAR4% + 6 × DAR6% + 8 × DAR8%) / (DAR0% + DAR2% + DAR4% + DAR6% + DAR8%).

Table 1

Table 2

Table 3

Example

[0059] Inhibition of Antibody-Drug Conjugates on the In Vitro Proliferation of Tumor Cells Human-derived tumor cells in the logarithmic growth phase were collected, digested with trypsin, resuspended in fresh complete medium, counted, adjusted to an appropriate concentration, and added to a 96-well cell culture plate at 100 μL / well. The cell culture plate was placed in a 37 °C, 5% CO2 incubator and incubated overnight. The next day, different concentrations of the ADC sample to be tested (maximum final concentration 1 μM, and 4-fold serial dilutions) or buffer control were added to the corresponding wells of the cell culture plate, and incubation was continued in the CO2 incubator for 120 hours. After equilibration to room temperature, the test plate was analyzed using the CellTiter Glo assay kit (Promega, G7558) and a multifunctional enzyme-labeling instrument (Enspire 2300, PerkinElmer) to detect the luminescence readings. The cell inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1 - (RLUADC - RLUblank) / (RLUbuffer - RLUblank)) × 100%, wherein, RLU represents Relative Luminescence Units. XLFit was used to plot the pharmacodynamic inhibition rate curve and calculate the IC 50 value (see Tables 4 and 5). The experimental results show that the ADCs tested exhibit excellent inhibitory activity against multiple tumor cell lines with different target expression levels, all of which are significantly better than the control ADCs. [Table 4] [Table 5]

Example

[0060] In vivo pharmacodynamic assay of antibody-drug conjugates in a subcutaneous xenograft tumor model of nude mice bearing human-derived tumor cells Human-derived tumor cells in the logarithmic growth phase were collected, dissociated, counted, resuspended in serum-free medium, and subcutaneously inoculated into female BALB / c nude mice at 100 μL / mouse to establish a xenograft tumor model. After the tumors grew to a measurable size, the major and minor diameters of each tumor were measured using calipers, and the tumor volume was calculated according to the following formula: V = (a × b 2 ) / 2 (where "a" represents the major diameter of the tumor and "b" represents the minor diameter of the tumor). When the tumor volume reached approximately 150 mm 3 , the mice were randomly grouped according to tumor volume and body weight. Solvent control (physiological saline) or different doses of ADC were injected into nude mice with tumors via the tail vein. The major and minor diameters of the tumors were measured twice a week; the tumor volumes were counted in each group, and the body weights of the animals were recorded. In the efficacy tests of MH-ADC1 and control ADC1 in a human lung cancer NCI-H1975 subcutaneous xenograft tumor model, the dosing regimen was once a week by tail vein injection, and the results of the test are shown in Figure 1. In the efficacy tests of MH-ADC2-1 and control ADC2 in a human lung cancer Calu-6 subcutaneous xenograft tumor model, on the day of grouping, the drug was administered once via the tail vein, and the results of the test are shown in Figure 2. In the efficacy tests of MH-ADC2-1 and control ADC2 in a human colorectal cancer HCT116 subcutaneous xenograft tumor model, the drug was administered once via the tail vein on the day of grouping and on the 11th day after grouping, respectively, and the results of the test are shown in Figure 3. The above test results indicate that the tested ADCs have a strong in vivo growth inhibitory effect in a subcutaneous xenograft tumor model of human-derived tumor cells in nude mice, are superior to the control ADCs at the same dose, and in particular, the antitumor efficacy of MH-ADC1 and MH-ADC2-1 is significantly better than the antitumor efficacy of the control ADCs with similar DAR values and the same dosing doses.

Claims

1. An antibody-drug conjugate represented by Formula I, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, wherein Ab-(L-D) ａ Formula I where Ab is an antibody such as a monoclonal antibody or its antigen-binding fragment; L is L １ -L ２ -L ３ -L ４ ; and 【Chemical 1】 L １ The carbonyl carbon terminus of ２ is connected to L １ and the alkyl carbon terminus of L is connected to Ab; n1 is an integer selected from 1 to 5; Preferably, n1 is 1, 2, or 3; n2 is an integer selected from 1 to 5; Preferably, n2 is 1 or 2; X １ is either a bond or O; X ２ is either a bond or O; 【Chemical Formula 2】 L ２ The amino terminus of L １ is connected to L ２ and the carbonyl terminus of L ３ is connected to L n3 is an integer selected from 1 to 5; Preferably, n3 is 1, 2, or 3; More preferably, n3 is 2; L ３ is an amino acid residue or a peptide residue consisting of 2 to 10 amino acid residues, and the amino terminus of L ３ is linked to L ２ and the carbonyl terminus of L ３ is linked to L ４ ; [Chemical 3] L ４ The amino terminus of L ３ is connected to L ４ and the carboxyl terminus of L is connected to D [Chemical Formula 4] The O-terminus of D is connected to L4, R １ is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, and halogenated C1-C6 alkyl; R ２ is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, and halogenated C1-C6 alkyl; R ３ is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, and halogenated C1-C6 alkyl; a is any number between 1 and 10.

2. L １ is 【Chemical Formula 5】 The antibody-drug conjugate according to Claim 1, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, selected from the group consisting of L １ The carbonyl carbon terminus of L is connected to L2, and the １ alkyl carbon terminus of L is connected to Ab. Preferably, L １ is [[Chemical Formula 6]] selected from the group consisting of; L １ The carbonyl carbon terminus of L is linked to L2, and the alkyl carbon terminus of L １ is linked to Ab.

3. L ３ is a peptide residue consisting of 2 to 4 (preferably 4) amino acid residues, with the amino terminus linked to L ２ and the carbonyl terminus linked to L ４ and preferably the amino acid is selected from the group consisting of glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably the amino acid is selected from the group consisting of glycine and phenylalanine, the antibody-drug conjugate according to claim 1 or 2, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, wherein Preferably, L ３ is glycine - glycine - phenylalanine - glycine (Gly - Gly - Phe - Gly), the amino terminus is connected to L ２ and the carbonyl terminus is connected to L ４ ; [Chemical Formula 7] The amino terminus is linked to L ２ and the carbonyl terminus is linked to L ４ .

4. R １ is an antibody-drug conjugate according to any one of claims 1 to 3, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl, wherein Preferably, R １ is C1-C6 alkyl; More preferably, R １ is methyl; or R ２ is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl; Preferably, R ２ is a halogen; More preferably, R ２ is fluorine; or, R ３ is selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl; Preferably, R ３ is C1-C6 alkyl; More preferably, R ３ is methyl.

5. L is 【Chemical 8】 【Chem.】 The antibody-drug conjugate according to any one of Claims 1 to 4, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, selected from the group consisting of The left carbon terminus of L is connected to Ab, and the right carbon terminus of L is connected to D, 【Chemical Formula 9】 L-D, as a whole, 【Chemical Formula 10】 【Chem.】

6. The antibody-drug conjugate represented by Formula I is selected from the group consisting of the following structural formulas. The antibody-drug conjugate according to any one of Claims 1 to 5, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate. 【Chemical Formula 11】 【Chem.】 (Wherein, Ab and a are defined as in Claim 1)

7. The antibody-drug conjugate according to any one of Claims 1 to 6, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, wherein each a is independently any number between 1 and 8, Preferably, each a is independently any number between 3 and 8; More preferably, each a is independently any number between 3 and 6.

8. The antibody-drug conjugate according to any one of Claims 1 to 7, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, wherein Ab is an anti-B7-H3 antibody or its antigen-binding fragment. Preferably comprising the anti-B7-H3 antibody or antigen-binding fragment thereof, (a) Three heavy chain variable region (VH) complementarity determining regions (CDRs) described below: (i) The CDR1 sequence contained in the VH shown in SEQ ID NO: 8, or A VH CDR1 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of one or two amino acids) compared to the CDR1 sequence contained in the VH; (ii) The CDR2 sequence contained in the VH shown in SEQ ID NO: 8, or a VH CDR2 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of one or two amino acids) compared to the CDR2 sequence contained in the VH; and (iii) The CDR3 sequence contained in the VH shown in SEQ ID NO: 8, or a VH CDR3 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of one or two amino acids) to the CDR3 sequence contained in the VH; And / or (b) Three light chain variable region (VL) CDRs described below: (iv) The CDR1 sequence contained in the VL shown in SEQ ID NO: 12, or a VL CDR1 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of one or two amino acids) to the CDR1 sequence contained in the VL; (v) The CDR2 sequence contained in the VL shown in SEQ ID NO: 12, or a VL CDR2 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of one or two amino acids) compared to the CDR2 sequence contained in the VL; and (vi) The CDR3 sequence contained in the VL shown in SEQ ID NO: 12, or a VL CDR3 having a sequence in which one or several amino acids are substituted, deleted or added (for example, substitution, deletion or addition of one or two amino acids) to the CDR3 sequence contained in the VL; Preferably, the substitution (or substituted) described in any one of (i) to (vi) is a conservative substitution; Preferably, CDR1, CDR2, and CDR3 included in the heavy chain variable region (VH), and / or CDR1, CDR2, and CDR3 included in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system, Preferably, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the anti-B7-H3 antibody or its antigen-binding fragment are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the anti-B7-H3 antibody or its antigen-binding fragment are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, More preferably, the amino acid sequence of the heavy chain variable region of the anti-B7-H3 antibody or its antigen-binding fragment is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region of the anti-B7-H3 antibody or its antigen-binding fragment is shown in SEQ ID NO: 12, Most preferably, the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown in SEQ ID NO: 2, according to the method of claim 1.

9. An antibody-drug conjugate according to any one of claims 1 to 8, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, and optionally one or more pharmaceutical excipients, a pharmaceutical composition comprising the same.

10. In the manufacture of a medicament for the treatment and / or prevention of a disease, the use of an antibody-drug conjugate according to any one of claims 1 to 8, its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the pharmaceutical composition according to claim 9, wherein Preferably, the disease is a disease associated with abnormal B7-H3 expression or function; Preferably, the disease is cancer or an autoimmune disease; More preferably, the disease is selected from the group consisting of lung cancer (e.g., undifferentiated lung cancer, small cell lung cancer, non-small cell lung cancer), esophageal cancer, gastric cancer, liver cancer, melanoma, prostate cancer, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, thyroid cancer, sarcoma, cholangiocarcinoma, glioblastoma, and neuroblastoma.

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