Antibody drug conjugate and application thereof

Abstract

The invention relates to an antibody drug conjugate and application thereof, and particularly provides an antibody drug conjugate shown as a formula I or a stereoisomer, a prodrug, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate of the antibody drug conjugate, Ab-(L-D) a.

Description

Antibody-drug conjugates and their applications

[0001] This disclosure is based on and claims priority to an application with CN application number 202210889771.5 and filing date July 27, 2022. The disclosure content of this CN application is hereby introduced into this disclosure as a whole. Technical Field

[0002] The present disclosure relates to the field of medical technology, and in particular to antibody-drug conjugates and applications thereof. Background Art

[0003] Antibody-drug conjugates (ADCs) are a new type of therapeutic macromolecule that combines the powerful lethality of small molecule drugs with the highly specific targeting properties of antibodies. Leveraging the specificity of antibodies, they can precisely deliver drugs to tumor sites and / or tumor cells, avoiding damage to normal cells in the body and thus reducing adverse reactions during treatment. ADCs consist of three components: an antibody, a toxin, and a linker. When the antibody portion of an ADC specifically binds to a specific antigen on the surface of a tumor cell, the tumor cell membrane indents inward, forming an endosome that engulfs the ADC. This process is known as internalization. The antibody portion of the ADC, or linker, is then degraded by enzymes within the cell, releasing the toxin, which then begins to kill the tumor cells.

[0004] B7-H3 (B7 homolog 3 protein), also known as CD276 and B7RP-2, is an immune checkpoint molecule and a member of the B7 family of immunomodulatory proteins. B7-H3 is expressed at low levels in normal tissues but is highly expressed in various solid tumors, such as lung cancer, head and neck cancer, esophageal cancer, ovarian cancer, breast cancer, prostate cancer, and melanoma. It is associated with poor prognosis in many tumors. Targeting B7-H3 may be an effective strategy for treating cancers with high B7-H3 expression. Currently, several B7-H3-targeted therapies are under development, including monoclonal antibodies, bispecific antibodies, CAR-T, and antibody-drug conjugates (ADCs), but no approved drugs have yet been marketed. Among them, antibody-drug conjugates use specific antibodies against B7-H3 to target small molecule toxins (payload) into tumor cells where the target is highly expressed. While retaining the tumor-killing properties of small molecule toxins, they selectively reduce the off-target toxic side effects of small molecule toxins. This has great potential in improving the benefit-risk ratio of anti-tumor treatment and is expected to bring greater benefits to patients.

[0005] Public content

[0006] The present disclosure provides an antibody-drug conjugate represented by Formula I, or its stereoisomers, prodrugs, pharmaceutically acceptable salts, or pharmaceutically acceptable solvates. This antibody-drug conjugate exhibits excellent inhibitory activity against multiple tumor cell lines with varying target expression levels and exhibits strong in vivo proliferation inhibition in a nude mouse subcutaneous xenograft model of human tumor cells, suggesting broad application prospects.

[0007] To this end, in the first aspect of the present disclosure, the present disclosure provides an antibody drug conjugate represented by Formula I or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate,

[0008] Ab-(LD) a

[0009] Formula I

[0010] in:

[0011] Ab is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof;

[0012] L is L 1 -L 2 -L 3 -L 4 ;

[0013] L 1 Selected from And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab;

[0014] n1 is an integer selected from 1 to 5;

[0015] n2 is an integer selected from 1 to 5;

[0016] X 1 Selected from direct bond, O;

[0017] X 2 Selected from direct bond, O;

[0018] L 2 Select from direct keys, And L 2 for When L 2 The amino terminus of L 1 Connected to the carbonyl end and L 3 connected;

[0019] n3 is an integer selected from 1 to 5;

[0020] L 3is selected from amino acid residues or peptide residues consisting of 2-10 amino acid residues, and the amino terminus is connected to L 2 Connected to the carbonyl end and L 4 connected;

[0021] L 4 for And its amino terminus is connected to L 3 Connected, the carbon end is connected to D;

[0022] D is And its O end and L 4 connected;

[0023] R 1 Selected from hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl;

[0024] R 2 Selected from hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl;

[0025] R 3 Selected from hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl;

[0026] a is any value between 1 and 10.

[0027] In some embodiments, n1 is selected from 1, 2, and 3. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5.

[0028] In some embodiments, n2 is selected from 1 and 2. In some embodiments, n2 is 1. In some embodiments, n2 is 2. In some embodiments, n2 is 3. In some embodiments, n2 is 4. In some embodiments, n2 is 5.

[0029] In some embodiments, n3 is selected from 1, 2, and 3. In some embodiments, n3 is 2. In some embodiments, n3 is 1. In some embodiments, n3 is 3. In some embodiments, n3 is 4. In some embodiments, n3 is 5.

[0030] In some embodiments, L 1 Selected from And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab.

[0031] In some embodiments, L1 Selected from And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab.

[0032] In some embodiments, L 1 for And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab.

[0033] In some embodiments, L 1 for And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab.

[0034] In some embodiments, L 1 for And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab.

[0035] In some embodiments, L 3 It is a peptide residue consisting of 2-4 (preferably 4) amino acid residues, and the amino terminus is connected to L 2 Connected to the carbonyl end and L 4 Preferably, the amino acid is selected from glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and more preferably, the amino acid is selected from glycine and phenylalanine.

[0036] In some embodiments, L 3 Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), and the amino terminus is connected to L 2 Connected to the carbonyl end and L 4 connected.

[0037] In some embodiments, L 3 for The amino terminus is connected to L 2 Connected to the carbonyl end and L 4 connected.

[0038] In some embodiments, R 1 Selected from hydrogen, halogen, C1-C6 alkyl.

[0039] In some embodiments, R 1 Selected from hydrogen, halogen, C1-C4 alkyl.

[0040] In some embodiments, R1 Selected from C1-C6 alkyl.

[0041] In some embodiments, R 1 In some embodiments, R 1 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. 1 Selected from methyl, ethyl, and n-propyl.

[0042] In some embodiments, R 1 Selected from fluorine, chlorine, bromine and iodine.

[0043] In some embodiments, R 1 Selected from fluorine, chlorine, and bromine.

[0044] In some embodiments, R 1 It is a methyl group.

[0045] In some embodiments, R 2 Selected from hydrogen, halogen, C1-C6 alkyl.

[0046] In some embodiments, R 2 Selected from hydrogen, halogen, C1-C4 alkyl.

[0047] In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. 2 Selected from methyl, ethyl, and n-propyl.

[0048] In some embodiments, R 2 In some embodiments, R 2 In some embodiments, R 2 Selected from fluorine, chlorine, and bromine.

[0049] In some embodiments, R 2 In some embodiments, R 2 It is a methyl group.

[0050] In some embodiments, R 3 Selected from hydrogen, halogen, C1-C6 alkyl.

[0051] In some embodiments, R 3 Selected from hydrogen, halogen, C1-C4 alkyl.

[0052] In some embodiments, R 3 In some embodiments, R 3 Selected from fluorine, chlorine, and bromine.

[0053] In some embodiments, R 3 Selected from C1-C6 alkyl.

[0054] In some embodiments, R 3 Selected from C1-C4 alkyl.

[0055] In some embodiments, R 3 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. 3 Selected from methyl, ethyl, and n-propyl.

[0056] In some embodiments, R 3 It is a methyl group.

[0057] In some embodiments, L is selected from:

[0058] The left carbon end is connected to Ab, and the right carbon end is connected to D.

[0059] In some embodiments, D is selected from In some embodiments, D is

[0060] In some embodiments, D is

[0061] In some embodiments, LD as a whole is selected from:

[0062] In some embodiments, the antibody drug conjugate of Formula I is selected from:

[0063] wherein Ab and a are as defined in the present disclosure.

[0064] In some embodiments, each a is independently any value between 1 and 8 (e.g., about 1, about 1.5, about 2, about 2.5, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8, or such as 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).

[0065] In some embodiments, each a is independently any number between 3-8.

[0066] In some embodiments, each a is independently any number between 3-6.

[0067] In some embodiments, each a is independently any number between 3.5 and 4.5 (e.g., about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5), for example, each a is independently about 3.9 or about 4.0.

[0068] In some embodiments, the Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof.

[0069] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof comprises:

[0070] (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0071] (i) a VH CDR1 having the sequence of the CDR1 contained in the VH as shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VH;

[0072] (ii) a VH CDR2 having the sequence of the CDR2 contained in the VH as shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR2 contained in the VH; and

[0073] (iii) a VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH;

[0074] and / or

[0075] (b) the following three light chain variable region (VL) CDRs:

[0076] (iv) a VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VL;

[0077] (v) a VL CDR2 having the sequence of the CDR2 contained in the VL as shown in SEQ ID NO: 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and

[0078] (vi) VL CDR3 having the sequence of the CDR3 contained in VL as shown in SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in VL.

[0079] In some embodiments, the substitution described in any one of (i)-(vi) is a conservative substitution.

[0080] In some embodiments, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering systems.

[0081] In some embodiments, the amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 of the anti-B7-H3 antibody or 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 the light chain variable region CDR1, CDR2, and CDR3 of the anti-B7-H3 antibody or 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 in the present disclosure are defined using the Kabat method.

[0082] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-B7-H3 antibody or 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 antigen-binding fragment thereof is shown in SEQ ID NO:12.

[0083] 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.

[0084] In a second aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and optionally one or more pharmaceutical excipients. In some embodiments, the antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate is present in an effective amount.

[0085] In the third aspect of the present disclosure, the present disclosure provides use of the aforementioned antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease, wherein the disease is a disease associated with abnormal B7-H3 expression or function.

[0086] In some embodiments, the disease is cancer or an autoimmune disease.

[0087] In some embodiments, the disease is selected from lung cancer (such as 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, bile duct cancer, glioblastoma, neuroblastoma.

[0088] In a fourth aspect of the present disclosure, the present disclosure provides the aforementioned antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the aforementioned pharmaceutical composition, for use in treating and / or preventing a disease associated with abnormal B7-H3 expression or function.

[0089] In some embodiments, the disease is cancer or an autoimmune disease.

[0090] In some embodiments, the disease is selected from lung cancer (such as 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, bile duct cancer, glioblastoma, neuroblastoma.

[0091] In a fifth aspect of the present disclosure, the present disclosure provides a method for treating and / or preventing a disease, comprising administering to a subject in need thereof an effective amount of the aforementioned antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, or the aforementioned 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.

[0092] In some embodiments, the disease is selected from lung cancer (such as 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, bile duct cancer, glioblastoma, neuroblastoma.

[0093] In a sixth aspect of the present disclosure, the present disclosure provides a composition for treating and / or preventing a disease, comprising the aforementioned antibody drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate. 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 lung cancer (such as 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, bile duct cancer, glioblastoma, neuroblastoma.

[0094] In the seventh aspect of the present disclosure, the present disclosure provides a drug for treating and / or preventing a disease, which comprises the aforementioned antibody drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate 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 lung cancer (such as 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, bile duct cancer, glioblastoma, neuroblastoma.

[0095] Definition of terms

[0096] In this disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0097] In the present disclosure, the term "pharmaceutically acceptable salt" refers to (i) salts formed by an acidic functional group present in the compounds provided herein with a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-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-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts. and (ii) salts formed by basic functional groups present in the compounds provided herein with appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

[0098] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic compound with a suitable base to provide a pharmaceutically acceptable cation.

[0099] In the present disclosure, the term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compound of the present disclosure. Prodrugs become active compounds only under biological conditions through this reaction, or they have no or only low activity in their unreactive form. Prodrugs can generally be prepared using known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, 5th edition).

[0100] When the stereoisomers in the compounds described in the present disclosure are specifically designated as (R)- or (S)-isomers in the chemical name, it should be understood that the predominant configuration is the (R)-isomer or the (S)-isomer, respectively. Any asymmetric carbon atom may exist in the (R)-, (S)-, or (R, S)-configuration, preferably in the (R)- or (S)-configuration.

[0101] In this disclosure, "solvate" or "solvate" are used interchangeably to refer to a compound that exists in combination with a solvent molecule. The combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; for example, methanol or ethanol may form an "alcoholate," which may also be stoichiometric or non-stoichiometric. The term "solvate" as used herein refers to a solid form, i.e., a compound in solution in a solvent, which may be solvated but is not a solvate as the term is used herein.

[0102] For the purposes of this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These substances, in addition to the active ingredients, have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, dissolution enhancement, and sustained-release control. They are important components that may affect the quality, safety, and efficacy of drugs. Based on their source, they can be categorized as natural, semi-synthetic, and fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0103] In the present disclosure, the pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. The pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present disclosure or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

[0104] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0105] In the present disclosure, the term "subject" includes humans and non-human animals. Exemplary human subjects include human subjects suffering from a disease (e.g., a disease described herein) (referred to as a patient) or normal individuals. The term "non-human animal" in the present disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0106] As used herein, the term "effective amount" refers to that amount of a compound which, when administered, will relieve to some extent one or more of the symptoms of the condition being treated.

[0107] In this disclosure, the term "antibody" is used in its broadest sense, including complete monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, as long as they have the desired biological activity. In this disclosure, "antibody" and "immunoglobulin" can be used interchangeably.

[0108] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. Monoclonal antibodies have high specificity for a single determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized without contamination by other antibodies. The modifier "monoclonal" herein indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by a specific method.

[0109] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies, that is, a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, and the remaining portion is identical or homologous to another type, class, or subclass of antibody, as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0110] Monoclonal antibodies used in the present disclosure can be produced by many methods. For example, monoclonal antibodies for the present disclosure can be obtained by hybridoma methods using cells from many species (including mice, hamsters, rats and people) (see, for example, Kohler et al., 1975, Nature, 256:495), or obtained by recombinant DNA technology (see, for example, US 4,816,567), or 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).

[0111] In this disclosure, unless otherwise expressly stated, the descriptions “each independently selected from” and “each independently selected from” used throughout this document are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, or that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0112] In the present disclosure, "n1 is an integer selected from 1-5" means that n1 is selected from 1, 2, 3, 4, and 5. Other similar definitions can be understood with reference to the above content.

[0113] In this disclosure, the term "direct bond" means that the groups on both sides are directly connected, for example, L 1 for If X 1 is a direct key, then L 1 for Other similar definitions can be understood by referring to the above content.

[0114] In this disclosure, “L 1 Selected from And L 1 The carbonyl carbon end and L 2 connected, and the alkyl carbon end is connected to Ab", where L 1 The term "carbonyl carbon end" or "alkyl carbon end" is used to describe the 1 The connection relationship with other groups, specifically, L 1 The "carbonyl carbon end" refers to the carbonyl group located at L 1 The carbonyl carbon end at the connection site on the right side, and the "alkyl carbon end" refers to the carbonyl carbon end at the L 1 The alkyl carbon end at the connection site on the left side. Other similar definitions can be understood with reference to the above content.

[0115] In this disclosure, L 3 The amino terminus of L 2Connected to the carbonyl end and L 4 connected, but if L 2 is a direct bond, then those skilled in the art will understand that L 3 The amino terminus will be correspondingly 1 Connected to the carbonyl end, the carbonyl end is still connected to L 4 Other similar situations can be understood by referring to the above content.

[0116] In the present disclosure, the term "amino acid residue" refers to the incomplete amino acid structure remaining after the amino group of an amino acid loses a hydrogen and the carboxyl group loses a hydroxyl.

[0117] In this disclosure, L 3 is selected from amino acid residues or peptide residues consisting of 2-10 amino acid residues, wherein the types of the 2-10 amino acids may be the same or different from each other. 3 is a peptide residue consisting of 4 amino acid residues, and the amino acids are selected from glycine and phenylalanine, then L 3 It can be a peptide residue of glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), specifically it can be

[0118] Throughout this disclosure, substituents of the disclosed compounds are disclosed by group class or range. It is specifically noted that this disclosure includes every independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl groups.

[0119] In the present disclosure, the term "C1-C6 alkyl" refers to a straight-chain or branched alkyl group containing 1 to 6 carbon atoms, including, for example, "C1-C3 alkyl" or "C1-C4 alkyl", methyl, ethyl, etc. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0120] In the present disclosure, the term "C1-C4 alkyl" refers to a straight-chain or branched alkyl group containing 1-4 carbon atoms, including, for example, "C1-C3 alkyl", methyl, ethyl, etc. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0121] Halogen refers to fluorine, chlorine, bromine or iodine.

[0122] In the present disclosure, the term "halogenated C1-C6 alkyl" refers to a group in which one or more hydrogen atoms in any of the above alkyl groups (e.g., C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, etc.) are replaced by halogen (preferably fluorine or chlorine), for example, monofluoromethyl, difluoroethyl, trifluoromethyl, etc.

[0123] In the present disclosure, the term "deuterated C1-C6 alkyl" refers to a group in which one or more hydrogen atoms in any of the above alkyl groups (e.g., C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, etc.) are replaced by deuterium atoms, for example, monodeuteriomethyl, dideuteriomethyl, trideuteriomethyl, etc.

[0124] Those skilled in the art will understand that in the antibody-drug conjugates disclosed herein, the antibody is linked to the linker-drug (linker-payload) via -S-, and that -S- is not an additional external thiol group, but rather a thiol group contained in the antibody itself after the antibody is reduced and the disulfide bonds are opened.

[0125] In the present disclosure, the drug-antibody ratio (DAR) refers to the number of drug molecules coupled to the antibody (e.g., a in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugates 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 coupled to each antibody. "a is any value between 1-10" means that a can be any integer selected from 1-10 (including endpoints 1 and 10), or any decimal selected from 1-10, such as 3.9 or 4.0. At the same time, it will be understood by those skilled in the art that even if the same preparation method is used, the DAR values ​​of the antibody-drug conjugates prepared in different batches are not necessarily exactly the same, for example, they can fluctuate within a range of no more than 0.5.

[0126] In the present disclosure, the term "about" is understood to mean 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. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about."

[0127] In the present disclosure, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the essential properties of the protein / polypeptide comprising the 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 substitutions in which an amino acid residue is substituted with an amino acid residue having a similar side chain, such as a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace 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, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference). Beneficial effects

[0128] 1. The ADC disclosed herein has excellent inhibitory activity against multiple tumor cell lines with different target expression levels.

[0129] 2. The ADC disclosed in the present invention has a strong in vivo proliferation inhibitory effect in a nude mouse subcutaneous transplant tumor model of human tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] The drawings described herein are used to provide a further understanding of the present disclosure and constitute a part of this application. The illustrative embodiments of the present disclosure and their descriptions are used to explain the present disclosure and do not constitute an improper limitation of the present disclosure. In the drawings:

[0131] Figure 1 shows the tumor growth curves of each drug-treated group in the human lung cancer NCI-H1975 subcutaneous xenograft tumor model in nude mice;

[0132] Figure 2 shows the tumor growth curves of each drug-treated group in the human lung cancer Calu-6 subcutaneous xenograft tumor model in nude mice;

[0133] Figure 3 shows the tumor growth curves of each drug-dosing group in the human colorectal cancer HCT116 subcutaneous transplanted tumor model in nude mice. DETAILED DESCRIPTION

[0134] The present disclosure will be further described below by way of specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art may make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure. Reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0135] In the following examples, the abbreviations of some drugs and their corresponding structural formulas are shown below:

[0136] DXd:

[0137] MC-GGFG-DXd:

[0138] Among them, MC-GGFG-DXd can be prepared by referring to Example 58 of CN104755494A.

[0139] In the following examples, the antibodies used are all known antibodies, and the sequences of the antibodies are shown below:

[0140] The CDRs (underlined) are defined below using the Kabat method.

[0141] Antibody P7-C05-H4L3 sequence (see WO2021244590A1):

[0142] >Heavy chain

[0143] From left to right, the unshaded portion represents the heavy chain variable region sequence (SEQ ID NO: 8), and the shaded portion represents the heavy chain constant region sequence. Within the heavy chain variable region sequence, the underlined portions, from left to right, represent the heavy chain CDR1 (SEQ ID NO: 5), heavy chain CDR2 (SEQ ID NO: 6), and heavy chain CDR3 (SEQ ID NO: 7).

[0144] >Light chain

[0145] From left to right, the unshaded portion represents the light chain variable region sequence (SEQ ID NO: 12), and the shaded portion represents the light chain constant region sequence. Within the light chain variable region sequence, the underlined portions, from left to right, represent the light chain CDR1 (SEQ ID NO: 9), light chain CDR2 (SEQ ID NO: 10), and light chain CDR3 (SEQ ID NO: 11).

[0146] Control ADC2 antibody (Ifinatamab) sequence (see Reference Example 1 in CN 104755494A, i.e., M30-H1-L4 antibody):

[0147] >Heavy chain

[0148] From left to right, the unshaded portion represents the heavy chain variable region sequence (SEQ ID NO: 16), and the shaded portion represents the heavy chain constant region sequence. Within the heavy chain variable region sequence, the underlined portions, from left to right, represent the heavy chain CDR1 (SEQ ID NO: 13), heavy chain CDR2 (SEQ ID NO: 14), and heavy chain CDR3 (SEQ ID NO: 15).

[0149] >Light chain

[0150] From left to right, the unshaded portion represents the light chain variable region sequence (SEQ ID NO: 20), while the shaded portion represents the light chain constant region sequence. Within the light chain variable region sequence, the underlined portions, from left to right, represent the light chain CDR1 (SEQ ID NO: 17), light chain CDR2 (SEQ ID NO: 18), and light chain CDR3 (SEQ ID NO: 19).

[0151] The present disclosure is further explained below with reference to specific examples. Unless otherwise specified, all reaction raw materials can be purchased commercially.

[0152] Example 1 Preparation of Linker-Drug

[0153] 1.1 Preparation of Compound II-12-a

[0154] first step

[0155] Compound 1a (50.00 g, 135.90 mmol) and 2-hydroxybenzyl acetate (45.00 g, 271.70 mmol) were added sequentially to a 1 L three-necked flask. After nitrogen displacement, anhydrous tetrahydrofuran (500 mL) was added. The reaction system was placed in an ice bath, and anhydrous p-toluenesulfonic acid (2.30 g, 13.40 mmol) was added. The reaction was stirred and continued for 2 hours under ice bath. Water (1 L) and ethyl acetate (1 L) were added to the reaction solution, and the layers were separated. The organic phase was washed sequentially with aqueous sodium bicarbonate solution, water, and aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 1b (27.00 g) in a yield of 42%.

[0156] MS-ESI calculated value [M+Na] + 497, measured value: 497.

[0157] Step 2

[0158] Compound 1b (17.00 g, 35.86 mmol) was dissolved in N,N-dimethylacetamide (170 mL), and 1,8-diazobisspiro[5.4.0]undec-7-ene (2.78 g, 18.29 mmol) was added. The mixture was reacted at room temperature under nitrogen for 1 hour. 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 then added to the reaction mixture. The mixture was stirred at room temperature for 2.5 hours. 2-Methyltetrahydrofuran (175 mL) and saturated aqueous sodium chloride solution (260 mL) were added to the reaction solution, and the organic phase was extracted and separated. The aqueous phase was further extracted with 2-methyltetrahydrofuran (100 mL×2). The organic phases were combined and washed once with 10% citric acid (90 mL), three times with saturated sodium bicarbonate (175 mL×3), and once with saturated brine (100 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to about 50 mL. Isopropanol (120 mL) was added, and the mixture was further concentrated to about 50 mL. Isopropanol (300 mL) was added, and the temperature was raised to 60°C and stirred for 1 hour. The solution was then cooled to 0-5°C and stirred for 2 hours. The solid was collected by filtration and rinsed with 5°C isopropanol to obtain compound 1d (19.00 g). Yield: 78%.

[0159] Step 3

[0160] Compound 1d (11.50 g, 15.65 mmol) was dissolved in a mixture of tetrahydrofuran (230 mL) and water (115 mL). 1,8-diazabicyclo[5.4.0]undec-7-ene (10.70 g, 70.39 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. The reaction system was extracted five times with methyl tert-butyl ether (100 mL x 5). The aqueous phase was concentrated to obtain a yellow oil, which was dissolved in isopropanol (70 mL). This solution was then added dropwise to methyl tert-ether (700 mL). A white solid precipitated and was collected by filtration. The solid was dissolved in ethanol and concentrated under reduced pressure to obtain compound 1e (9.50 g) as a white foamy solid in a 93% yield.

[0161] Step 4

[0162] Compound 1e (1.00 g, 1.53 mmol) was dissolved in a mixture of acetonitrile (10 mL) and water (20 mL), and 1f (0.54 g, 1.75 mmol) was added. The mixture was stirred at room temperature under nitrogen for 5 hours. Water (20 mL) was added to the reaction system, and the pH was adjusted to 3-4 with 0.5N aqueous hydrochloric acid. The mixture was extracted four times with a mixed solvent of isopropanol / dichloromethane (v:v = 4:1, 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford a yellow oil. The oil was dissolved in a mixed solvent of dichloromethane / methanol (v:v = 4:1, 5 mL). The solution was added dropwise to methyl tert-butyl ether (100 mL), resulting in the precipitation of a white solid, which was collected by filtration to afford compound 1g (850 mg), in a 90% yield.

[0163] MS-ESI calculated value [M+Na] + 639, measured value: 639.

[0164] Step 5

[0165] Compound 1h (12.20 g, 46.39 mmol) was weighed into a three-necked flask (500 mL). After nitrogen replacement, the reaction flask was placed in an ice bath and anhydrous N,N-dimethylformamide (120 mL) was added. Imidazole (15.77 g, 232.00 mmol) and triethylsilyl chloride (27.97 g, 185.56 mmol) were added to the reaction system in sequence. After stirring for ten minutes, 4-dimethylaminopyridine (5.66 g, 46.39 mmol) was added and the reaction was continued at 0°C with stirring for 3 hours. The reaction system was diluted with methyl tert-butyl ether (1.20 L), and then water (1.2 L) was added for separation. The aqueous phase was extracted with methyl tert-butyl ether (1.2 L), and the organic phases were combined. The organic phase was washed once more with water (1.20 L), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to obtain crude product 1i (16.00 g).

[0166] Step 6

[0167] Crude product 1i (16.00 g, 32.59 mmol) and Lawesson's reagent (13.18 g, 32.59 mmol) were weighed into a 1 L three-necked flask. Anhydrous toluene (600 mL) was added. After nitrogen displacement, the reaction system was heated to 125°C and refluxed for 5 hours. The reaction solution was concentrated to obtain a residue, which was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to obtain crude product 1j (16.00 g).

[0168] Step 7

[0169] Crude product 1j (16 g) was dissolved in anhydrous tetrahydrofuran (640 mL). Triethylamine trihydrofluoride (11.93 g, 74.00 mmol) was added dropwise under ice-cooling. After complete addition, the mixture was slowly returned to room temperature with stirring. After reacting overnight at room temperature, the reaction system was diluted with ethyl acetate (1 L). The layers were separated, and the organic phase was washed with saturated brine (500 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to provide compound 1k (6.38 g), with a three-step yield of 49%.

[0170] Step 8

[0171] 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-methylphenol (15 mL) were placed in a 500 mL three-necked flask. Anhydrous toluene (300 mL) was added. After nitrogen displacement, the reaction system was heated to 120°C in an oil bath for 32 hours. The reaction system was cooled to room temperature and the insoluble solid was collected by filtration. The crude product was slurried with methyl tert-butyl ether (60 mL) for 1 hour, filtered, and dried to obtain compound 1m (3.0 g) in a yield of 51%.

[0172] Step 9

[0173] Compound 1m (2.30 g, 4.67 mmol) was weighed into a 500 mL single-necked bottle, and 6N aqueous hydrochloric acid (230 mL) was added. The reaction system was heated to 110°C and refluxed for 12 hours. The reaction system was hot filtered to remove insoluble matter, and the filtrate was concentrated under reduced pressure to obtain the crude product 1n (a set of diastereomers). Preparative HPLC purification afforded compounds 1n-P1 (412 mg, the less polar isomer by TLC) and 1n-P2 (495 mg, the more polar isomer by TLC) in a yield of 34%.

[0174] Step 10

[0175] 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 added to a 10 mL single-necked bottle in sequence. 2,4,6-trimethylpyridine (60 mg, 0.49 mmol) was then added dropwise to the mixture. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction solution was added dropwise to methyl tert-butyl ether (60 mL). Solids precipitated and were collected by filtration. The solids were dissolved in a mixed solvent of dichloromethane / methanol (V:V=6:1, 30 mL) and concentrated to obtain a crude product. The crude product was purified by liquid phase preparative separation to obtain compound II-12-a (33 mg) in a yield of 35%.

[0176] MS-ESI calculated value [M+H] + 1050, measured value: 1050.

[0177] 1H NMR (400MHz, DMSO-d6) δ8.65(t,J=6.4Hz,1H),8.60(d,J=8.8Hz,1H),8.28(t,J=5.6Hz,1H) ,8.10(d,J=8.0Hz,1H),8.06(t,J=6.0Hz,1H),7.99(t,J=5.6Hz,1H),7.79(d,J=10.8Hz,1H ),7.78(s,1H),7.28-7.14(m,5H),6.97(s,1H),5.90(d,J=16.4Hz,1H),5.70-5,60(m,1H), 5.51(s,1H),5.46(d,J=2.8Hz,1H),5.30(d,J=19.6Hz,1H),4.67(d,J=6.4Hz,1H),4.51-4.4 1(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=14.0,4.8Hz,1H),2.80-2.71(m,1H),2.37(s,3H),2.26-2.15(m,2H),2.08(t,J= 7.6Hz,2H),1.94-1.82(m,2H),1.54-1.37(m,4H),1.25-1.10(m,2H),0.85(t,J=7.6Hz,3H).

[0178] 1.2 Preparation of compound II-1-a

[0179] first step

[0180] 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. The mixture was stirred at room temperature under nitrogen atmosphere for 16 hours. This was designated as solution A and set aside.

[0181] Take a 50mL three-necked flask, add N-hydroxysuccinimide (3.46g, 30.08mmol) and N,N-dimethylformamide (10mL), cool to 0℃, add trifluoroacetic anhydride (6.31g, 30.08mmol) dropwise, stir for 0.5 hours, then add 2,4,6-trimethylpyridine (5.46g, 45.10mmol), continue stirring for 0.5 hours after the addition is complete, and add the reaction solution dropwise to the above solution A. After the addition is complete, stir at room temperature for 16 hours. The reaction system is added with 1N hydrochloric acid to adjust the pH to 2-3, extracted with dichloromethane (50mL), and the organic phase is washed three times with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue is purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain compound 2b (1.20g), yield: 51%.

[0182] Step 2

[0183] 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. The mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction solution was concentrated under reduced pressure and directly purified by preparative HPLC to give compound 2c (0.30 g) in a yield of 61%.

[0184] Step 3

[0185] To a 10 mL single-necked flask, compound 1n-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 added sequentially. 2,4,6-trimethylpyridine (60 mg, 0.49 mmol) was then added dropwise to the mixture. After the addition was complete, the mixture was stirred at room temperature for 3 hours. The reaction solution was added dropwise to methyl tert-butyl ether (60 mL). A solid precipitated and was collected by filtration. The solid was dissolved in a dichloromethane / methanol mixed solvent (V:V = 6:1, 30 mL) and concentrated to obtain a crude product. The crude product was purified by liquid phase preparative HPLC to obtain compound II-1-a (16 mg) in a yield of 17%.

[0186] MS-ESI calculated value [M+H] + 1052, measured value: 1052.

[0187] 1H NMR (400MHz, DMSO-d6) δ8.65(t,J=6.8Hz,1H),8.61(d,J=9.2Hz,1H),8.28(t,J=4. 4Hz,1H),8.14-8.06(m,2H),8.06(t,J=5.6Hz,1H),7.82(d,J=11.2Hz,1H),7.79(s ,1H),7.28-7.13(m,5H),6.99(s,2H),5.92(d,J=16.8Hz,1H),5.70-5,60(m,1H),5 .53(d,J=8.0Hz,1H),5.48(d,J=4.8Hz,1H),5.37(d,J=19.6Hz,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),3.1 9-3.08(m,1H),3.06-2.97(m,1H),2.80-2.70(m,1H),2.70-2.65(m,1H),2.39(s,3 H),2.35-2.28(m,3H),2.27-2.18(m,2H),1.94-1.82(m,2H),0.85(t,J=7.2Hz,3H).

[0188] 1.3 Preparation of Compound II-3-a

[0189] first step

[0190] 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. The mixture was stirred at room temperature under nitrogen for 16 hours. Water (250 mL) was added to the reaction solution, and the aqueous phase was extracted with dichloromethane / isopropanol (v:v = 4:1, 200 mL x 3). The organic phases were combined and concentrated under reduced pressure to obtain crude product 3b (12.00 g).

[0191] Step 2

[0192] Crude product 3b (4.00 g, 31.70 mmol) was dissolved in N,N-dimethylformamide (60 mL), and di(p-nitrobenzene) carbonate (16.40 g, 53.95 mmol) was added. The temperature was raised to 30°C, and N,N-diisopropylethylamine (4.18 g, 32.40 mmol) was added. The reaction mixture was stirred for 2 hours. The reaction solution was cooled to room temperature, and water (600 mL) was added. The aqueous phase was extracted with dichloromethane (300 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 0-100%) to obtain compound 3c (2.40 g), with a two-step yield of 13%.

[0193] Step 3

[0194] Compound 3c (400 mg, 1.14 mmol) was dissolved in a mixture of acetonitrile (8 mL) and water (16 mL). Compound 1e (760 mg, 1.13 mmol) was added and stirred at room temperature under nitrogen for 16 hours. The reaction solution was directly purified by preparative HPLC to afford compound 3d (109 mg) in a 15% yield.

[0195] Step 4

[0196] To a single-necked flask (10 mL) were added compound 1n-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) in sequence, and then 2,4,6-trimethylpyridine (84 mg, 0.69 mmol) was added dropwise to the mixture. The reaction was stirred at room temperature for 3 hours. The reaction solution was added dropwise to methyl tert-butyl ether (75 mL), and a solid precipitated. The solid was collected by filtration and dissolved in a dichloromethane / methanol mixed solvent (V:V=6:1, 30 mL). The residue was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to obtain compound II-3-a (45 mg) in a yield of 35%.

[0197] MS-ESI calculated value [M+H] + 1068.4, measured value: 1068.9.

[0198] 1H NMR (400MHz, DMSO-d6) δ8.68(t,J=6.8Hz,1H),8.62(d,J=9.2Hz,1H),8.31(t,J=5.6Hz,1H),8.12(d,J=8.0Hz,1H),7.99(t,J=5.6Hz,1H),7.78(s,1H ),7.77(d,J=10.8Hz,1H),7.38(t,J=6.0Hz,1H),7.27-7.13(m,5H),6.99( s,2H),5.90(d,J=16.4Hz,1H),5.70-5,61(m,1H),5.51(s,1H),5.46(d,J= 4.4Hz,1H),5.29(d,J=19.6Hz,1H),4.67(d,J=6.4Hz,1H),4.51-4.41(m,1 H),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=14.0,4.4Hz,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=7.2Hz,3H).

[0199] 1.4 Preparation of Compound II-2-a

[0200] first step

[0201] Compound 4a (2.00 g, 7.52 mmol) and β-alanine (0.67 g, 7.52 mmol) were dissolved in acetonitrile (20 mL). The mixture was cooled to 0°C and N,N-diisopropylethylamine (1.94 g, 15.03 mmol) was added. After the addition, the mixture was returned to room temperature and stirred for 16 hours. Water (100 mL) was added to the reaction solution, which was then extracted with dichloromethane / isopropanol (v:v = 4:1, 250 mL x 10). The organic phases were combined and concentrated to obtain a residue, which was then purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain compound 4b (2.00 g).

[0202] Step 2

[0203] 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. The mixture was stirred for 30 minutes after addition. N-hydroxysuccinimide (0.95 g, 8.25 mmol) was then added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was filtered and directly purified by preparative HPLC to obtain compound 4c (530 mg) in a yield of 21%.

[0204] Step 3

[0205] Compound 4c (0.50 g, 1.48 mmol) was dissolved in a mixture of acetonitrile (8 mL) and water (16 mL), and 1e (0.98 g, 1.50 mmol) was added. The mixture was stirred at room temperature under nitrogen for 16 hours. The reaction solution was directly subjected to liquid-phase preparative separation and purification to afford compound 4d (42 mg) in a 4% yield.

[0206] Step 4

[0207] To a single-necked flask (10 mL) were added compound 1n-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) in sequence. 2,4,6-trimethylpyridine (32 mg, 0.26 mmol) was then added dropwise to the mixture. The reaction was allowed to react at room temperature for 3 hours. The reaction solution was added dropwise to methyl tert-butyl ether (30 mL). A solid precipitated and was collected by filtration. The solid was dissolved in a dichloromethane / methanol mixed solvent (V:V = 6:1, 30 mL) and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC to obtain compound II-2-a (13 mg) in a yield of 26%.

[0208] MS-ESI calculated value [M+H] + 1079.4, measured value: 1079.9.

[0209] 1H NMR (400MHz, DMSO-d6) δ8.68(t,J=6.8Hz,1H),8.63(d,J=8.8Hz,1H),8.31(t,J=6.0Hz,1H),8.18(t,J=7.2Hz,1H),8.10(d,J=7.2Hz,1H),8.03(t,J=5 .6Hz,1H),7.98(t,J=5.6Hz,1H),7.82(d,J=10.8Hz,1H),7.79(s,1H),7.28 -7.13(m,5H),6.98(s,2H),6.70(s,1H),5.90(d,J=16.8Hz,1H),5.70-5,60 (m,1H),5.56-5.45(m,2H),5.36(d,J=19.6Hz,1H),4.68(d,J=6.8Hz,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=7.2Hz,3H).

[0210] 1.5 Preparation of Compound II-12-b

[0211] first step

[0212] Under nitrogen protection and 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). A solution of N,N-diisopropylethylamine (17.10 mg, 0.0133 mmol) in N,N-dimethylformamide (0.4 mL) was added dropwise, followed by a solution of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (24.20 mg, 0.064 mmol) in N,N-dimethylformamide (0.6 mL). After the addition was complete, the mixture was kept at 0°C for 1 hour. The reaction solution was purified to obtain compounds II-12-a (16 mg) and II-12-b (19 mg), with a combined yield of 64%.

[0213] II-12-b:

[0214] MS-ESI calculated value [M+H] +1050.4, measured value: 1050.3.

[0215] 1 H NMR (400MHz, DMSO-d6) δ8.71-8.61 (m, 2H), 8.31 (t, J = 4.8Hz, 1H), 8.12 (d,J=7.2Hz,1H),8.07(t,J=6.0Hz,1H),8.00(t,J=5.2Hz,1H),7.82(dd,J=10.8,2.0Hz,1H),7.80(s,1H),7.28-7.13(m,6H),6.99(s,2H),6.70(br s,1H),5.92(d,J=16.8Hz,1H),5.70-5,61(m,1H),5.52(d,J=17.2Hz,2H),5.37(d,J=19.6Hz,1H) ,4.68(d,J=6.8Hz,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=7.6Hz,2H),1.96-1.82(m,2H),1.52-1.39(m,4H),1.25-1.10(m,2H),0.85(t,J=7.6Hz,3H).

[0216] 1.6 Preparation of Compounds X and Y

[0217] first step

[0218] To a single-necked flask (25 mL) were added 1n (20 mg, 0.04 mmol) and a 1.2 mg / mL solution of glycolic acid in N,N-dimethylformamide (2.00 mL, 0.032 mmol) in sequence. The reaction system was cooled to 0 ° C. N,N-diisopropylethylamine (11 mg, 0.08 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphorus were added in sequence. To the mixture was added 1.2 mg / mL of glycolic acid in N,N-dimethylformamide (0.20 mL, 0.003 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1 mg). The reaction was continued for 1 hour, and the reaction system was diluted with ethyl acetate (80 mL). The organic phase was washed with 0.5N dilute hydrochloric acid (5 mL x 1), saturated sodium bicarbonate solution (10 mL x 2) and saturated brine (10 mL x 5), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative thin-layer chromatography (methanol:dichloromethane) to give compounds X (2 mg) and Y (5 mg). The total yield was 34%.

[0219] X:

[0220] MS-ESI calculated value [M+H] + 510, the actual measured value is 510.

[0221] 1 H NMR (400MHz, DMSO-d6) δ8.54(d,J=8.8Hz,1H),7.82(d,J=10.8Hz,1H),7.80(s,1H),5.92(d,J=16.8Hz,1H),5.69–5.60 (m,1H),5.53(d,J=20.4Hz,1H),5.52(d,J=16.4Hz,1H),5.35(d,J=20.0Hz,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=7.2Hz,3H).

[0222] Y:

[0223] MS-ESI calculated value [M+H] + 510, the actual measured value is 510.

[0224] 1H NMR (400MHz, DMSO-d6) δ8.54(d,J=9.2Hz,1H),7.81(d,J=11.2Hz,1H),7.80(s,1H) ,6.69(s,1H),5.92(d,J=16.8Hz,1H),5.69–5.62(m,1H),5.62–5.56(m,1H),5.55–5 .47(m,2H),5.33(d,J=20.0Hz,1H),4.16–3.98(m,2H),3.30–3.20(m,1H),3.16–3.0 7(m,1H),2.39(s,3H),2.26–2.15(m,2H),1.95–1.85(m,2H),0.86(t,J=7.2Hz,3H).

[0225] Example 2 Preparation and Detection of Antibody-Drug Conjugates

[0226] 1. General preparation method

[0227] Dialyze the antibody into 50mM PB buffer to obtain an antibody intermediate. Take an appropriate amount of the antibody intermediate and sequentially add a 10mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock solution and a 10mM diethylenetriaminepentaacetic acid (DTPA) stock solution. Add 50mM PB buffer to achieve a final antibody concentration of 5-10mg / mL, a TCEP:antibody molar ratio of (2.2-8.0):1.0, and a final DTPA concentration of 0.5-1.5mM. Mix thoroughly and place in a thermomixer at 25±2°C, 400rpm, and a reduction reaction time of 1.0-2.5 hours. After reduction, add an appropriate amount of 5mM linker-drug stock solution to each reaction system in an ice-water bath, achieving a linker-drug:antibody molar ratio of 4.5-12.0. If the purity of the conjugated product is low, consider adding DMSO and / or arginine hydrochloride. After thorough mixing, place in a thermomixer at 25 ± 2°C, 400 rpm, and allow the coupling reaction to proceed for 0.5 to 1.5 hours. After the coupling is complete, dialyze the ADC sample into a dialysate (10 mM or 20 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifuge tube to obtain the ADC stock solution, which is then aliquoted and stored at -80°C.

[0228] 1.1 Preparation of MH-ADC1

[0229] Antibody P7-C05-H4L3 was dialyzed into 50mM PB buffer (pH 7.4±0.2) to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was added to a 10mM stock solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a 10mM stock solution of diethylenetriaminepentaacetic acid (DTPA). 50mM PB buffer (pH 7.4±0.2) was then added to achieve a final antibody concentration of 10mg / mL, a TCEP to antibody molar ratio of 2.4:1.0, and a final DTPA concentration of 1mM. The mixture was thoroughly mixed and placed in a thermomixer for reduction at 25±2°C, 400 rpm, and 150 min. Linker-payload II-12-b was prepared in DMSO to a 5mM stock solution. After reduction, add an appropriate amount of 5 mM linker-drug II-12-b stock solution to the reaction system in an ice-water bath to achieve a linker-drug to antibody molar ratio of 8.0. After thorough mixing, place the reaction in a thermomixer at 25 ± 2°C, 400 rpm, and allow the coupling reaction to proceed for 60 min. After coupling, dialyze the resulting ADC sample into a dialysate (20 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifuge tube to obtain the MH-ADC1 stock solution, which is then aliquoted and stored at -80°C.

[0230] 1.2 Preparation of MH-ADC2

[0231] 1.2.1 Preparation of MH-ADC2-1

[0232] Antibody P7-C05-H4L3 was dialyzed into 50mM PB buffer (pH 7.4±0.2) to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was added sequentially to a 10mM stock solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a 10mM stock solution of diethylenetriaminepentaacetic acid (DTPA). 50mM PB buffer (pH 7.4±0.2) was then added to the reaction system to achieve a final antibody concentration of 10mg / mL, a TCEP to antibody molar ratio of 2.6:1.0, and a final DTPA concentration of 1mM. After thorough mixing, the mixture was placed in a thermomixer and reduced at 25±2°C, 400rpm, and 2h. Linker-drug II-1-a was prepared in DMSO to a 5mM stock solution. After reduction, an appropriate amount of the 5mM stock solution of linker-drug II-1-a was added to the reaction system in an ice-water bath to achieve a linker-drug to antibody molar ratio of 5.5. After thorough mixing, place in a thermomixer and allow the coupling reaction to proceed at 25 ± 2°C, 400 rpm, for 1 hour. After the coupling is complete, the resulting ADC sample is dialyzed into a dialysate (10 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifuge tube to obtain the MH-ADC2-1 stock solution, which is then aliquoted and stored at -80°C.

[0233] 1.2.2 Preparation of MH-ADC2-2

[0234] Antibody P7-C05-H4L3 was dialyzed into 50mM PB buffer (pH 7.4±0.2) to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was added sequentially to a 10mM stock solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a 10mM stock solution of diethylenetriaminepentaacetic acid (DTPA). 50mM PB buffer (pH 7.4±0.2) was then added to the reaction system to achieve a final antibody concentration of 5mg / mL, a TCEP to antibody molar ratio of 2.2:1.0, and a final DTPA concentration of 1mM. After thorough mixing, the mixture was placed in a thermomixer and reduced at 25±2°C, 400rpm, for 1h. Linker-drug II-1-a was prepared in DMSO to a 5mM stock solution. After reduction, an appropriate amount of the 5mM stock solution of linker-drug II-1-a was added to the reaction system in an ice-water bath to achieve a linker-drug to antibody molar ratio of 5.0. After thorough mixing, place in a thermomixer at 25 ± 2°C, 400 rpm, and allow the coupling reaction to proceed for 1 hour. After the coupling is complete, the resulting ADC sample is dialyzed into a 10 mM His / His-HCl dialysate (pH 6.0 ± 0.2) using an ultrafiltration centrifuge tube to obtain the MH-ADC2-2 stock solution, which is then aliquoted and stored at -80°C.

[0235] 1.2.3 Preparation of MH-ADC2-3

[0236] Antibody P7-C05-H4L3 was dialyzed into 50mM PB buffer (pH 7.7±0.2) to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was added sequentially to a 10mM stock solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a 10mM stock solution of diethylenetriaminepentaacetic acid (DTPA). 50mM PB buffer (pH 7.7±0.2) was then added to the reaction system to achieve a final antibody concentration of 10mg / mL, a TCEP to antibody molar ratio of 3.5:1.0, and a final DTPA concentration of 1mM. After thorough mixing, the mixture was placed in a thermomixer and reduced at 25±2°C, 400rpm, and 2h. Linker-drug II-1-a was prepared in DMSO to a 5mM stock solution. After reduction, an appropriate amount of the 5mM stock solution of linker-drug II-1-a was added to the reaction system in an ice-water bath to achieve a linker-drug to antibody molar ratio of 7.0. After thorough mixing, place in a thermomixer and allow the coupling reaction to proceed at 25 ± 2°C, 400 rpm, for 1 hour. After the coupling is complete, the resulting ADC sample is dialyzed into a dialysate (10 mM His / His-HCl, pH 6.0 ± 0.2) using an ultrafiltration centrifuge tube to obtain the MH-ADC2-3 stock solution, which is then aliquoted and stored at -80°C.

[0237] 1.3 Preparation of control ADC1

[0238] Antibody P7-C05-H4L3 was dialyzed into 50mM PB buffer (pH 7.4±0.2) to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was added sequentially to a 10mM stock solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a 10mM stock solution of diethylenetriaminepentaacetic acid (DTPA). 50mM PB buffer (pH 7.4±0.2) was then added to achieve a final antibody concentration of 10mg / mL, a TCEP to antibody molar ratio of 2.6:1.0, and a final DTPA concentration of 1mM. After thorough mixing, the mixture was placed in a thermomixer and reduced at 25±2°C, 400rpm, and 150min. The linker-drug MC-GGFG-DXd was prepared in DMSO to a 5mM stock solution. After reduction, add an appropriate amount of 5mM linker-drug MC-GGFG-DXd stock solution to the reaction system in an ice-water bath to achieve a linker-drug to antibody molar ratio of 8.0. After thorough mixing, place the reaction in a thermomixer at 25±2°C, 400 rpm, and allow the coupling reaction to proceed for 60 minutes. After coupling, dialyze the resulting ADC sample into a dialysate (20mM His / His-HCl, pH 6.0±0.2) using an ultrafiltration centrifuge tube to obtain a control ADC1 stock solution, which is then aliquoted and stored at -80°C.

[0239] 1.4 Preparation of control ADC2

[0240] The antibody Ifinatamab was dialyzed into 50mM PB buffer (pH 7.4±0.2) to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was added sequentially to a 10mM stock solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a 10mM stock solution of diethylenetriaminepentaacetic acid (DTPA). 50mM PB buffer (pH 7.4±0.2) was then added to achieve a final antibody concentration of 5mg / mL, a TCEP to antibody molar ratio of 2.4:1.0, and a final DTPA concentration of 1mM. After thorough mixing, the mixture was placed in a thermomixer and reduced at 25±2°C, 400rpm, and 2h. The linker-drug MC-GGFG-DXd was prepared in DMSO to a 5mM stock solution. After reduction, an appropriate amount of 1M arginine hydrochloride was added to achieve a final concentration of 50mM in the coupling reaction. In an ice-water bath, add an appropriate amount of DMSO and a 5mM linker-drug MC-GGFG-DXd stock solution to the reaction system, achieving a final DMSO concentration of 15% and a linker-drug to antibody molar ratio of 4.5. After thorough mixing, place the reaction mixture in a thermomixer at 25±2°C, 400 rpm, and allow the coupling reaction to proceed for 1 hour. Following coupling, dialyze the resulting ADC sample into a dialysate (10mM His / His-HCl, pH 6.0±0.2) using an ultrafiltration centrifuge tube to obtain a control ADC2 stock solution, which was aliquoted and stored at -80°C.

[0241] 2. Drug-antibody conjugate ratio detection of antibody-drug conjugates

[0242] The average drug-to-antibody ratio (DAR) of ADCs was determined using hydrophobic high-performance liquid chromatography (HIC-HPLC). After equilibration to room temperature and mixing, the ADC sample was diluted with ultrapure water to a target concentration of 5 mg / mL based on the protein concentration and mixed on a vortex mixer for 20 seconds. The diluted sample solution was pipetted into an insert and the sample vial was labeled with the sample number. After column equilibration, the sample was analyzed using an HPLC instrument (Agilent 1260Bio) using a hydrophobic chromatography column (TOSOH, TSKgel Butyl-NPR column (2.5), 4.6 mm x 35 mm). The main acquisition parameters are shown in Tables 1 and 2. Mobile phase A consisted of 20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0; mobile phase B consisted of 20 mM sodium phosphate, 25% isopropanol (v / v), pH 7.0. The liquid phase chromatogram was integrated, and the percentages of the ADCs unconjugated with drug and conjugated with two, four, six, and eight drugs (DAR0%, DAR2%, DAR4%, DAR6%, and DAR8%, respectively) were calculated based on the integration results. The average drug-antibody conjugation ratio was calculated using the following formula, as shown in Table 3. DAR value = (0 × DAR0% + 2 × DAR2% + 4 × DAR4% + 6 × DAR6% + 8 × DAR8%) / (DAR0% + DAR2% + DAR4% + DAR6% + DAR8%).

[0243] Table 1. Acquisition method parameters of MH-ADC1 and control ADC1

[0244] Table 2. Acquisition method parameters for MH-ADC2-1, MH-ADC2-2, MH-ADC2-3, and control ADC2

[0245] Table 3. Drug-Antibody Coupling Ratio (DAR) Test Results

[0246] Example 3 Inhibition of tumor cell proliferation in vitro by antibody-drug conjugates

[0247] Human tumor cells in the logarithmic growth phase were obtained, trypsinized, resuspended in fresh complete culture medium, counted, and adjusted to the appropriate concentration. The cells were then added to a 96-well cell culture plate at 100 μL / well. The cell culture plates were incubated in a 37°C, 5% CO2 incubator overnight. The next day, different concentrations of the ADC samples to be tested (maximum final concentration 1 μM, 4-fold serial dilution) or buffer control were added to the corresponding wells of the cell culture plates. After further incubation in a CO2 incubator for 120 hours, the test plates were equilibrated to room temperature, and luminescence readings were measured using the CellTiter Glo assay kit (Promega, G7558) and a multi-function microplate reader (Enspire 2300, PerkinElmer). The cell inhibition rate was calculated according to the following formula: Inhibition rate (%) = (1 – (RLU) ADC –RLU 空白 ) / (RLU 缓冲液 –RLU 空 白 ))×100%, where RLU represents relative luminescence units. XLFit was used to draw the drug inhibition rate curve and calculate IC 50 Values ​​(see Table 4 and Table 5).

[0248] Experimental results showed that the tested ADC had excellent inhibitory activity against multiple tumor cell lines with different target expression levels, and was significantly better than the control ADC.

[0249] Table 4. Inhibitory activity of ADC on proliferation of human tumor cells in vitro

[0250] Table 5. Inhibitory activity of ADC on proliferation of human tumor cells in vitro

[0251] Example 4 In vivo efficacy testing of antibody-drug conjugates in a nude mouse subcutaneous transplant model of human tumor cells

[0252] Human tumor cells in the logarithmic growth phase were digested and counted, then resuspended in serum-free culture medium and inoculated subcutaneously into female BALB / c nude mice. 100 μL was inoculated per animal to establish a nude mouse xenograft tumor model. After the tumor grew to a measurable range, the long and short diameters of each tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = (a × b 2 ) / 2, where a represents the long diameter of the tumor and b represents the short diameter of the tumor.

[0253] When the tumor volume reaches an average of 150 mm 3Around 200 mice were randomly divided into groups based on tumor volume and body weight. Tumor-bearing nude mice were injected via the tail vein with either a solvent control (normal saline) or different doses of ADC. Tumor length and short diameter were measured twice weekly, and tumor volume and body weight were recorded for each group.

[0254] The efficacy of MH-ADC1 and control ADC1 in the human lung cancer NCI-H1975 subcutaneous xenograft tumor model was tested with a weekly tail vein injection schedule. The test results are shown in Figure 1. The efficacy of MH-ADC2-1 and control ADC2 in the human lung cancer Calu-6 subcutaneous xenograft tumor model was tested with a single tail vein injection on the day of grouping. The test results are shown in Figure 2. The efficacy of MH-ADC2-1 and control ADC2 in the human colorectal cancer HCT116 subcutaneous xenograft tumor model was tested with a single tail vein injection on the day of grouping and 11 days after grouping. The test results are shown in Figure 3.

[0255] The above experimental results show that the tested ADCs all have strong in vivo proliferation inhibition effects in the human tumor cell subcutaneous transplant tumor model in nude mice, and are superior to the control ADC at the same dose. In particular, the anti-tumor efficacy of MH-ADC1 and MH-ADC2-1 is significantly better than that of the control ADC with similar DAR values ​​and the same dosage.

Claims

1. An antibody-drug conjugate of formula I, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, Ab-(LD) a Formula I in: 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 ; L 1 Selected from And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab; n1 is an integer selected from 1 to 5; Preferably, n1 is selected from 1, 2, and 3; n2 is an integer selected from 1 to 5; Preferably, n2 is selected from 1, 2; X 1 Selected from direct bond, O; X 2 Selected from direct bond, O; L 2 Select from direct keys, And L 2 for When L 2 The amino terminus of L 1 Connected to the carbonyl end and L 3 connected; n3 is an integer selected from 1 to 5; Preferably, n3 is selected from 1, 2, 3; More preferably, n3 is 2; L 3 is selected from amino acid residues or peptide residues consisting of 2-10 amino acid residues, and the amino terminus is connected to L 2 Connected to the carbonyl end and L 4 connected; L 4 for And its amino terminus is connected to L 3 Connected, the carbon end is connected to D; D is And its O end and L 4 connected; R 1 Selected from hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl; R 2 Selected from hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl; R 3 Selected from hydrogen, deuterium, halogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl; a is any value between 1 and 10.

2. The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate according to claim 1, wherein: L 1 Selected from And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab; Preferably, L 1 Selected from And L 1 The carbonyl carbon end and L 2 Connected, the alkyl carbon end is connected to Ab.

3. The antibody-drug conjugate or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate according to any one of claims 1 to 2, wherein: L 3 It is a peptide residue consisting of 2-4 (preferably 4) amino acid residues, and the amino terminus is connected to L 2 Connected to the carbonyl end and L 4 Preferably, the amino acid is selected from glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid, and aspartic acid, more preferably, the amino acid is selected from glycine and phenylalanine; Preferably, L 3 is selected from Gly-Gly-Phe-Gly, and the amino terminus is connected to L 2 Connected to the carbonyl end and L 4 connected; More preferably, L 3 for The amino terminus is connected to L 2 Connected to the carbonyl end and L 4 connected.

4. The antibody-drug conjugate or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate according to any one of claims 1 to 3, wherein: R 1 Selected from hydrogen, halogen, C1-C6 alkyl; Preferably, R 1 Selected from C1-C6 alkyl; More preferably, R 1 is methyl; Or, R 2 Selected from hydrogen, halogen, C1-C6 alkyl; Preferably, R 2 selected from halogen; More preferably, R 2 For fluorine; Or, R 3 Selected from hydrogen, halogen, C1-C6 alkyl; Preferably, R 3 Selected from C1-C6 alkyl; More preferably, R 3 It is a methyl group.

5. The antibody drug conjugate or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate according to any one of claims 1 to 4, wherein: L is selected from: The left carbon end is connected to Ab, and the right carbon end is connected to D; Alternatively, D is selected from Alternatively, LD as a whole is selected from:

6. The antibody-drug conjugate or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate according to any one of claims 1 to 5, wherein: The antibody drug conjugate represented by Formula I is selected from: wherein Ab and a are as defined in claim 1.

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

8. The antibody-drug conjugate or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate according to any one of claims 1 to 7, wherein: Ab is an anti-B7-H3 antibody or an antigen-binding fragment thereof; Preferably, the anti-B7-H3 antibody or antigen-binding fragment thereof comprises: (a) the following three heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having the sequence of the CDR1 contained in the VH as shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having the sequence of the CDR2 contained in the VH as shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having the sequence of the CDR3 contained in the VH as shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VH; and / or (b) the following three light chain variable region (VL) CDRs: (iv) a VL CDR1 having the sequence of the CDR1 contained in the VL as shown in SEQ ID NO: 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having the sequence of the CDR2 contained in the VL as shown in SEQ ID NO: 12, or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1 or 2 amino acid substitutions, deletions or additions) compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having the sequence of the CDR3 contained in the VL as shown in SEQ ID NO: 12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution described in any one of (i) to (vi) is a conservative substitution; Preferably, the CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2 and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia or IMGT numbering systems; Preferably, the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the anti-B7-H3 antibody or 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 the light chain variable regions CDR1, CDR2, and CDR3 of the anti-B7-H3 antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region of the anti-B7-H3 antibody or antigen-binding fragment thereof is as 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.

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

10. Use of the antibody-drug conjugate according to any one of claims 1 to 8, or its stereoisomer, prodrug, pharmaceutically acceptable salt or pharmaceutically acceptable solvate, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating and / or preventing a disease. 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 lung cancer (such as 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, bile duct cancer, glioblastoma, neuroblastoma.

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