Auristatin antibody-drug conjugate, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of medicinal chemistry, and particularly relates to an auristatin antibody-drug conjugate, a preparation method therefor, and use thereof. The antibody-drug conjugate has excellent DAR4 uniformity and drug activity, dynamic characteristics, and safety, and thus is suitable for preparing a drug for treating tumor-related diseases.

Description

Auristatin antibody-drug conjugates and preparation methods and applications thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefits and priority of Chinese patent application No. 2024101716205 filed on February 6, 2024, Chinese patent application No. 2024106290336 filed on May 20, 2024, Chinese patent application No. 2024113052826 filed on September 18, 2024, and Chinese patent application No. 2024118755141 filed on December 18, 2024, the entire contents of which are hereby incorporated by reference into this document in their entirety. Technical Field

[0003] The present invention belongs to the field of medicinal chemistry technology, and specifically relates to auristatin-type antibody-drug conjugates, their preparation methods, and uses. These antibody-drug conjugates exhibit excellent DAR4 homogeneity, pharmacological activity, and kinetic properties, making them suitable for the preparation of drugs for the treatment of tumor-related diseases. Background Art

[0004] Cancer is a major challenge worldwide, with an estimated 20 million new cases of cancer and 10 million cancer-related deaths in 2020. Advances in targeted therapies, including immunotherapy, biomarker-targeted therapies, and antibody-drug conjugates (ADCs), have revolutionized the treatment of certain cancers. MMAE is a potent antimitotic agent that inhibits tubulin polymerization. Although it has demonstrated strong antitumor activity in the clinic, its use is often hampered by severe toxicities such as myelosuppression and neuropathy. Currently, four MMAE-based ADCs have been approved by the US FDA, validating the benefits of MMAE payloads in oncology. To activate and release the therapeutic payload, the conjugate needs to be internalized into tumor cells and then processed by endogenous proteases. However, the internalization requirement limits the range of antigens that can be targeted by ADCs, as not all extracellular tumor-associated antigens are internalized.

[0005] Furthermore, many highly expressed antigens are secreted in the tumor stroma or localized on cancer-associated fibroblasts rather than on tumor cells themselves. Technologies that specifically activate MMAE in the tumor microenvironment through non-internalized pathways or stromal cell antigens will complement ADC therapy. Advances in drug delivery systems and ADC technology have revitalized the use of previously undruggable and potentially side-effect-prone toxins.

[0006] Antibody-drug conjugates (ADCs) are composed of a monoclonal antibody targeting a specific antigen and a small molecule cytotoxic drug linked via a linker. They combine the powerful anti-tumor effects of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs. ADC technology effectively overcomes the shortcomings of toxic drugs alone, such as poor targeting, significant side effects, a narrow therapeutic window, and limited tissue distribution. The powerful targeting capabilities of the antibody moiety enable the toxin to be effectively delivered to target tissues, thereby enhancing the therapeutic efficacy of these compounds. Summary of the Invention

[0007] The first aspect of the present invention provides an antibody drug conjugate of formula I or a pharmaceutically acceptable salt thereof: Ab-[-L1-L2-L3-L4-D] q Formula I

[0008] Wherein, Ab is an antibody portion or antigen-binding fragment; D is a drug molecule; -L1-L2-L3-L4- is a fragment connecting Ab and drug molecule D; q is selected from 2-9;

[0009] L1 is a linker portion connected to the antibody, which is selected from the following structures:

[0010] The end marked with an asterisk * of L1 is connected to Ab;

[0011] L2 is selected from the following fragments: wherein g and p are each independently selected from 0, 1, 2, 3, 4, 5; j is selected from 5, 6, 7, 8, 9; k is selected from 2, 3, 4, 5; and the end of L2 marked with an asterisk * is connected to L1;

[0012] L3 is a polypeptide fragment consisting of 2-4 amino acid residues, wherein the amino acid residues are selected from the following α-amino acid residues: glycine, phenylalanine, valine, alanine, asparagine, citrulline, lysine, serine, glutamic acid, and aspartic acid; and the -NH-terminus of L3 is connected to L2;

[0013] L4 is a chemical bond or L4 is selected from the following groups: -NH-CH2-, Wherein, R3 is selected from or R3 is selected from H, OH, CN, NH2, halogen atoms, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 haloalkoxy; said r is selected from 5, 6, 7, 8, 9; the -NH- end of L4 is connected to L3.

[0014] In any technical solution of formula I of the present invention, the drug molecule D is selected from camptothecin compounds, auristatin compounds, eribulin compounds, maytansine compounds, calicheamicin compounds, and anthramycin compounds.

[0015] In any technical solution of formula I of the present invention, the drug molecule D is an auristatin compound.

[0016] In any technical solution of formula I of the present invention, the drug molecule D is MMAE.

[0017] In any one of the technical solutions of formula I of the present invention, q is selected from 2-6, preferably 3-5, more preferably 3.5-4.5 or 4.0-4.5.

[0018] In any one of the technical solutions of formula I of the present invention, q is selected from 7-9, preferably 7.5-8.5, and more preferably 7.5-8.0.

[0019] In any technical solution of formula I of the present invention, g is selected from 0, 1, and 2.

[0020] In any technical solution of formula I of the present invention, p is selected from 1, 2, and 3.

[0021] In any technical solution of formula I of the present invention, k is selected from 3, 4, and 5, and preferably k is 4.

[0022] In any technical solution of formula I of the present invention, j is selected from 6, 7, and 8, and preferably j is 7.

[0023] In any technical solution of formula I of the present invention, j is selected from 7, p is 1 or 3, g is 1, and k is 4.

[0024] In any technical solution of formula I of the present invention, r is selected from 6, 7, and 8, and preferably r is 7.

[0025] In any technical solution of formula I of the present invention, Ab is selected from anti-Her2 antibody and anti-5T4 antibody. The anti-Her2 antibody is selected from trastuzumab and pertuzumab.

[0026] In any technical solution of formula I of the present invention, R3 is selected from H, F, Cl, methyl, and ethyl, and preferably R3 is H.

[0027] In any technical solution of formula I of the present invention, L2 is selected from the following fragments:

[0028] In any technical solution of formula I of the present invention, L3 is selected from a polypeptide fragment consisting of 2-4 residues of the following amino acids: glycine, phenylalanine, valine, alanine, citrulline, and asparagine; and the -NH-terminus of L3 is connected to L2.

[0029] In any technical solution of formula I of the present invention, L3 is selected from the following polypeptide fragments: -Val-Cit-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-Asn-; and the -NH-terminus of L3 is connected to L2.

[0030] In any technical solution of formula I of the present invention, L4 is selected from Its -NH-terminus is connected to L3.

[0031] In any technical solution of formula I of the present invention, -L1-L2-L3-L4- is selected from the following fragments:

[0032] In any technical solution of formula I of the present invention, -L1-L2-L3-L4-D is selected from the following fragments:

[0033] In one technical solution of Formula I of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the chemical structure described in Formula II:

[0034] wherein Ab, L3, g, j, p, and q have the same meanings as in Formula I or any of its technical solutions.

[0035] In any technical solution of formula II of the present invention, L3 can be selected from -Val-Cit-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-Asn-, and the -NH-terminus of L3 is connected to -CO-.

[0036] In any technical solution of formula II of the present invention, j is selected from 6, 7, and 8, and preferably j is 7.

[0037] In any technical solution of formula II of the present invention, g is selected from 0, 1, and 2, and preferably g is 1.

[0038] In any technical solution of formula II of the present invention, q is selected from 3-5, preferably 3.5-4.5.

[0039] In any technical solution of formula II of the present invention, q is selected from 7.5-8.5, preferably 7.5-8.0.

[0040] In any technical solution of formula II of the present invention, p is selected from 1, 2, and 3.

[0041] In any technical solution of Formula II of the present invention, Ab is selected from anti-Her2 antibody and anti-5T4 antibody.

[0042] In any technical solution of Formula II of the present invention, Ab is selected from trastuzumab and pertuzumab.

[0043] In a technical solution of formula II of the present invention, j is 7, g is 1, p is selected from 1 or 3; q is 3.5-4.5, L3 is selected from -Val-Cit-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-Asn-, and the -NH- end of L3 is connected to -CO-.

[0044] In one technical solution of Formula II of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the chemical structure described in Formula II-1:

[0045] wherein q is 3.5-4.5; the Ab

[0046] In one technical solution of Formula I of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the chemical structure described in Formula III-1 or III-2:

[0047] Wherein, Ab, L3, q, and k have the same meanings as in Formula I or any technical solution thereof.

[0048] In any technical solution of formula III-1 or formula III-2 of the present invention, q is selected from 3-5, preferably 3.5-4.5.

[0049] In any technical solution of formula III-1 or formula III-2 of the present invention, q is selected from 7.5-8.5, preferably 7.5-8.0.

[0050] In any technical solution of formula III-1 or formula III-2 of the present invention, k is selected from 3, 4, and 5; preferably, k is 4.

[0051] In any technical solution of Formula III-1 or Formula III-2 of the present invention, Ab is selected from anti-Her2 antibody and anti-5T4 antibody.

[0052] In any technical solution of Formula III-1 or Formula III-2 of the present invention, Ab is selected from trastuzumab and pertuzumab.

[0053] In any technical solution of formula III-1 or formula III-2 of the present invention, L3 is selected from -Val-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, and -Ala-Ala-Asn-; and the -NH-terminus of L3 is connected to the adjacent -CO-.

[0054] In any technical solution of formula III-1 or formula III-2 of the present invention, k is 4, q is 3.5-4.5, L3 is selected from -Val-Cit-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-Asn-, and the -NH- end of L3 is connected to -CO-.

[0055] In any technical solution of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody.

[0056] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0057] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0058] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0059] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having a heavy chain variable region as shown in SEQ ID NO: 4.

[0060] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having a heavy chain as shown in SEQ ID NO: 5.

[0061] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having a light chain variable region as shown in SEQ ID NO:9.

[0062] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having a light chain as shown in SEQ ID NO: 10.

[0063] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having a heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO: 9.

[0064] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-5T4 antibody having a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10.

[0065] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is the antibody PF6263507.

[0066] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody, which comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0067] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody, which comprises a heavy chain variable region as shown in SEQ ID NO: 14.

[0068] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody comprising a heavy chain as shown in SEQ ID NO: 15.

[0069] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody, which comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0070] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody comprising a light chain variable region as shown in SEQ ID NO: 19.

[0071] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody comprising a light chain as shown in SEQ ID NO: 20.

[0072] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody, which comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0073] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody, which comprises a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 19.

[0074] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is an anti-Her2 antibody, which comprises a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20.

[0075] In any one of the technical solutions of Formula I, Formula II, Formula II-1, Formula III-1, and Formula III-2 of the present invention, the Ab is Trastuzumab.

[0076] The second aspect of the present invention provides the following compounds or pharmaceutically acceptable salts thereof:

[0077] A third aspect of the present invention provides the following fragment:

[0078] The fourth aspect of the present invention provides use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention in the preparation of a drug for treating cancer.

[0079] The fifth aspect of the present invention provides use of the compound according to the second aspect of the present invention or a pharmaceutically acceptable salt thereof in preparing the antibody-drug conjugate according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0080] The sixth aspect of the present invention provides use of the structural fragment described in the third aspect of the present invention in preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof described in the first aspect of the present invention.

[0081] The seventh aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention.

[0082] The eighth aspect of the present invention provides a method for treating cancer, which comprises administering an effective amount of the compound of the first aspect of the present invention or a pharmaceutically acceptable salt thereof to a patient in need, or administering an effective amount of the pharmaceutical composition of the seventh aspect of the present invention to a patient in need.

[0083] It should be noted that for Ab-[-L1-L2-L3-L4-D] q Those skilled in the art will understand that the S atom marked with an asterisk (*) at the end of L1 in the present invention refers to the S atom generated by the connection between L1 and the thiol group contained in the Ab itself. That is, the S atom between L1 and Ab is not a separate external sulfur atom. Even if the asterisked S atom is not shown or described in L1 of the present invention, it is generated when Ab is coupled with L1-L2-L3-L4-D, and the resulting ADC molecule also contains this S atom. Therefore, the following structures should have the same meaning, and the other structures should be understood similarly.

[0084] In the present invention, the antibody PF6263507 has a sequence as shown in the following Table S1 defined by the Kabat numbering system.

[0085] Table S1 Sequence of antibody PF6263507

[0086] More specifically, the antibody PF6263507 has a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10:

[0087] In the present invention, the anti-Her2 antibody may be Trastuzumab, which has a sequence defined by the Kabat numbering system as shown in Table S2 below.

[0088] Sequence of the S2 antibody Trastuzumab

[0089] More specifically, the antibody Trastuzumab has a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20:

[0090] The present invention provides excellent technical effects: The compounds of the first and second aspects of the present invention, or their pharmaceutically acceptable salts, exhibit excellent cancer-inhibiting effects and acceptable toxicity. The preparation method of the present invention also offers significant technical advantages. When preparing ADCs, the proportion of the DAR4 component in the ADCs obtained by the present invention is significantly superior to that of other existing technologies, resulting in improved uniformity.

[0091] Definitions and Explanations of Terms

[0092] Unless otherwise indicated, the terms used herein have their ordinary meanings in the art. A particular term or phrase should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0093] The term "drug molecule," also known as "load," "drug payload," or "payload," refers to a substance that has the potential to prevent or treat a disease. The drug in an antibody-drug conjugate is typically a cytotoxic drug, a chemical molecule that has the potential to disrupt the normal growth of tumor cells.

[0094] The term "linker" refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to the drug.

[0095] The term "drug-linker conjugate" is also referred to as payload-linker conjugate, linker-payload conjugate, and has the same meaning in the present invention.

[0096] In the present invention, an ADC drug conjugate with a DAR3 refers to an ADC molecule with a drug-antibody ratio of 3, i.e., one antibody is linked to three cargo molecules; an ADC drug conjugate with a DAR4 refers to an ADC molecule with a drug-antibody ratio of 4, i.e., one antibody is linked to four cargo molecules; and "DAR value" represents the average DAR of the resulting drug conjugate ADC.

[0097] In the present invention, H refers to the hydrogen element, that is, a type of atom with a nucleus having one proton, which includes three isotopes: protium (P), deuterium (D), and tritium (T). H also represents an atom of the hydrogen element.

[0098] In the present invention, the term "independently selected" means that when multiple substituents exist simultaneously between different variable groups and between the same substituent, each variable group can select the same or different selected ranges or options.

[0099] In the present invention, the "halogen" or "halogen atom" refers to fluorine, chlorine, bromine, and iodine; the "halo" refers to a group formed when one or more hydrogen atoms in a substituent are replaced by a halogen atom.

[0100] The term "alkyl" refers to a straight or branched hydrocarbon group in which carbon atoms and carbon atoms are connected to hydrogen atoms by single bonds. 1- 4 or C 1-6 Alkyl; C 1-4 "Alkyl" means a straight or branched chain alkyl group having 1 to 4 carbon atoms; C 1-4 Specific examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl, preferably methyl, ethyl, n-propyl, and isopropyl; "C 1-6 "Alkyl" means a straight or branched chain alkyl group having 1 to 6 carbon atoms. 1-6 Specific examples of alkyl groups include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl.

[0101] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a halogen atom. 1-4 Halogenated alkyl, C 1-6 Halogenated alkyl. C 1-4 Specific examples of haloalkyl include, but are not limited to, monofluoromethyl, monochloromethane, difluoromethyl, dichloromethane, trifluoromethyl, trichloromethyl, and tribromomethyl.

[0102] The term "alkoxy" refers to an alkyl-O-, i.e., a substituent formed by replacing the hydrogen atom in -OH with an alkyl group. Alkyl groups are as described above, and alkoxy groups include C 1-4 Alkoxy, C 1-6 Alkoxy; C 1-4 Specific examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, preferably methoxy, ethoxy, isooxy; C 1-6 Specific examples of alkoxy groups include C 1-4 In addition to the specific examples of alkoxy, n-pentyloxy, neopentyloxy and n-hexyloxy are also included.

[0103] The term "haloalkoxy" refers to a substituent obtained by replacing one or more hydrogen atoms of an alkoxy group with a halogen atom. 1-4 Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, 2,2,2-trichloroethoxy.

[0104] The term "hydroxy-C 1-4 "Alkyl" refers to C 1-4 A substituent obtained by replacing one or more hydrogen atoms of an alkyl group with a hydroxyl group. 1-4Specific examples of the alkyl group include hydroxymethyl and hydroxyethyl.

[0105] The term "cycloalkyl" refers to a cyclic, saturated monocyclic structure formed by single bonds between carbon atoms. Specific examples of 3-6 membered cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; specific examples of 5-6 membered cycloalkyl groups include, but are not limited to, cyclopentyl and cyclohexyl.

[0106] The term "heterocycloalkyl" refers to a substituent group obtained by replacing one or more carbon atoms in the annular backbone of a cycloalkyl group with one or more heteroatoms or heteroatoms; the heteroatoms or heteroatoms are generally selected from N, N(O), O, S, S(O), S(O)2. Specific examples of the 5-6 membered heterocycloalkyl group include, but are not limited to, tetrahydropyrrolyl, tetrahydrofuranyl, piperidinyl, and piperazinyl.

[0107] In the present invention, the "natural amino acids" refer to the following amino acids with α-configuration: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile I), proline (Pro), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), and histidine (His).

[0108] The term "unnatural amino acid" refers to amino acids other than the natural amino acids, such as citrulline.

[0109] The term "optionally" means that the substituent may be substituted by other substituents or may not be substituted by other substituents.

[0110] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product resulting, directly or indirectly, from combination of the specified ingredients in the specified amounts. Those skilled in the art can modify the actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention so as to achieve the desired therapeutic response in an amount effective for a particular patient, composition, and route of administration.

[0111] The term "pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, especially mammals.

[0112] The term "excipient" generally refers to a carrier, diluent and / or vehicle required to formulate an effective pharmaceutical composition.

[0113] The term "effective amount" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, which is a sufficient amount of the compound to treat the disorder at a reasonable benefit / risk ratio applicable to any medical treatment and / or prevention.

[0114] The term "pharmaceutically acceptable salt" refers to salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio.

[0115] In the present invention, a chemical bond in a substituent is marked with When , it indicates that the substituent is connected to the adjacent group or structural fragment at this position. The presence of a dash "-" in the substituent structure indicates the point of attachment for the substituent, for example, -CH3 is connected through a C atom. Indicates the absolute configuration of a stereocenter, i.e., R or S configuration.

[0116] When a substituent's bond crosses two atoms in a ring, the substituent may be bonded to any atom in the ring. The substituent R can be substituted at any position on the benzene ring.

[0117] The "isomers" described in the present invention include geometric isomers and stereoisomers, such as atropisomers, cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures and other mixtures thereof, all of which fall within the scope of the present invention. The term "enantiomer" refers to stereoisomers that are mirror images of each other. The term "tautomer" refers to a type of functional group isomer that has different hydrogen attachment points due to the displacement of one or more double bonds, for example, a ketone and its enol form are keto-enol tautomers. The term "diastereomer" refers to stereoisomers in which the molecule has two or more chiral centers and is not a mirror image of the other molecules. The term "cis-trans isomers" refers to different spatial configurations in which double bonds or single bonds of ring carbon atoms in a molecule cannot rotate freely. The term "atropisomers" refers to stereoisomers that can be separated because single bond rotation is hindered or rotates very slowly.

[0118] Stereoisomers of the compounds of the present invention can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. For example, one enantiomer of a compound of the present invention can be prepared by asymmetric catalysis or chiral auxiliary derivatization. Alternatively, a compound of a single stereoconfiguration can be obtained from a mixture by chiral resolution techniques. Alternatively, they can be prepared directly using chiral starting materials. Separation of optically pure compounds in the present invention is typically accomplished by preparative chromatography using chiral chromatographic columns to achieve the purpose of separating chiral compounds.

[0119] In the present invention, the unit of solution concentration M represents mol / L, and nM represents mmol / L. In the embodiments of the present invention, the component ratios of the eluent used in silica gel column chromatography are all volume ratios.

[0120] In the present invention, the compound Vedotin refers to the following compound, which can be purchased or prepared by referring to the published document US7659241B2;

[0121] The chemical abbreviations used in the present invention and the chemical names they refer to are as follows:

[0122] Figures in the specification

[0123] Figure 1: SEC spectrum of drug conjugate ADC1;

[0124] Figure 2: SEC spectrum of drug conjugate ADC2;

[0125] Figure 3: SEC spectrum of drug conjugate ADC3;

[0126] Figure 4: SEC spectrum of drug conjugate ADC4;

[0127] Figure 5: HIC spectrum of drug conjugate ADC1;

[0128] Figure 6: HIC spectrum of drug conjugate ADC2;

[0129] Figure 7: HIC spectrum of drug conjugate ADC3;

[0130] Figure 8: HIC spectrum of drug conjugate ADC4;

[0131] Figure 9: Inhibition rate-concentration relationship diagram of drug conjugates ADC1 and ADC2 in the SKGT-4 model;

[0132] Figure 10: Inhibition rate-concentration relationship diagram of drug conjugates ADC3 and ADC4 in the SK-BR-3 model;

[0133] Figure 11: Inhibition rate-concentration relationship diagram of drug conjugates ADC3 and ADC4 in the MDA-MB-486 model;

[0134] Figure 12: In vivo efficacy of drug conjugates ADC1 and ADC2;

[0135] Figure 13: In vivo efficacy of drug conjugates ADC3 and ADC4;

[0136] Figure 14: Mouse plasma stability study of drug conjugates ADC3 and ADC4;

[0137] Figure 15: Results of drug conjugate efficacy evaluation in JIMT-1 tumor-bearing mice. DETAILED DESCRIPTION

[0138] The present invention is further described below through specific preparation examples and biological experiments. However, it should be understood that these examples and biological experiments are for illustrative purposes only and should not be construed as limiting the present invention in any way. It will be apparent to those skilled in the art that, unless otherwise specified, the materials used are well known in the art and can be purchased commercially or obtained by those skilled in the art according to published literature or conventional methods. For example, the antibody trastuzumab was purchased from Sanyou Biopharmaceuticals (Shanghai) Co., Ltd. Unless otherwise specified, all reactions of the present invention were carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, using dry solvents. (i) Temperatures are expressed in degrees Celsius (°C), and operations were performed at room temperature, generally 15-35°C, preferably 20-30°C, and more preferably 20-25°C; (ii) Solvent removal was performed by rotary evaporation under reduced pressure, with the bath temperature not exceeding 60°C; (iii) Reaction progress was monitored by thin-layer chromatography (TLC); and (iv) the final product exhibited satisfactory H nuclear magnetic resonance spectroscopy (1H-NMR) and / or mass spectrometry (MS) data.

[0139] Test equipment:

[0140] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements are performed using a Bruker Neo 400M or Bruker Ascend 400 NMR instrument, using deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and / or deuterated chloroform (CDCl3) as the solvent, and tetramethylsilane (TMS) as the internal standard.

[0141] Liquid chromatography-mass spectrometry (LC-MS) was performed using a Shimadzu 2030Plus-LCMS2020 mass spectrometer, an Agilent 1260-6125B single quadrupole mass spectrometer, or a Shimadzu LCMS-2020 mass spectrometer. HPLC was performed using a Shimadzu LCMS-2020 or Agilent 1260 high-performance liquid chromatograph.

[0142] Preparative high-performance liquid chromatography was performed using a Shimadzu FRC-40 equipped with LC-20AP and PDA-20A (column: Synergi Max-RP, 150×30 mm, 4 μm) or a GILSON GX-281LC (columns: Boston Prime C18 150*30 mm*5 μm; YMC-Actus Triart C18 150*30 mm*5 μm; YMC-Triart PFP 150*30 mm*5 μm; YMC-Triart Phenyl 150*30 mm*5 μm).

[0143] The preparation and separation conditions for the payload-linker coupling compounds in the examples of the present invention are as follows: (Chromatographic column: Boston Prime C18 150*30mm*5μm; A: 0.225% FA in Waters; B: MeCN). Those skilled in the art can appropriately adjust the elution gradient according to different example compounds.

[0144] Example D9: Preparation of Compound D9

[0145] Step 1: Synthesis of compound D9-1

[0146] To a 100 mL single-necked flask, mono-tert-butyl succinate (1.12 g, 6.44 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.45 g, 6.44 mmol) were added and dissolved in N,N-dimethylformamide (15 mL). Diisopropylethylamine (0.01 mL, 0.09 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. Dipropargylamine (500 mg, 5.37 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (petroleum ether:ethyl acetate = 3:1) to obtain compound D9-1 (1.1 g, 82.2%).

[0147] LC-MS: 194.1[M-56+H] + .

[0148] Step 2: Synthesis of compound D9-2

[0149] D9-1 (249 mg, 1.0 mmol) and N,N-dimethylformamide (10 mL) were added to a 100 mL three-necked flask. 5-Bromo-2-methylthiopyrimidine (410 mg, 2 mmol), cuprous iodide (20 mg, 0.1 mmol), triphenylphosphine (52 mg, 0.2 mmol), bis(acetonitrile)palladium dichloride (26 mg, 0.1 mmol), and diisopropylamine (505 mg, 5 mmol) were then added at room temperature. The atmosphere was purged with nitrogen three times and stirred at 80°C for 2 h. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified on a silica gel column (petroleum ether:ethyl acetate = 3:1) to afford D9-2 (390 mg, 78.5%).

[0150] LC-MS:498.2[M+H] + .

[0151] Step 3: Synthesis of compound D9-3

[0152] D9-2 (390 mg, 0.78 mmol) was added to a 100 mL single-necked flask and dissolved in dichloromethane (8 mL). Meta-chloroperbenzoic acid (840 mg, 3.9 mmol) was slowly added to the resulting solution at 0°C. The reaction solution was slowly warmed to room temperature in an ice-water bath under nitrogen and stirred overnight. The reaction solution was placed in dry ice-ethanol solution (-78°C) and cooled with stirring. A large amount of white solid precipitated. The solid was quickly filtered while still cold, and the filtrate was collected. The filtrate was concentrated under reduced pressure in a 25°C water bath. The residue was purified on a silica gel column (petroleum ether:ethyl acetate = 1:1) to afford D9-3 (220 mg, 50.3%).

[0153] LC-MS: 506.1[M-56+H] + .

[0154] Step 4: Synthesis of compound D9-4

[0155] D9-3 (220 mg, 0.39 mmol) was added to a 50 mL single-necked flask and dissolved in dichloromethane (4 mL). Trifluoroacetic acid (1 mL) was added to the resulting solution. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was diluted with toluene (4 mL) and concentrated under reduced pressure. Purification was performed on a C18 column (20% MeCN, 80% 0.1% TFA in Water) to afford D9-4 (120 mg, 60.9%).

[0156] LC-MS: 506.1[M+H] + .

[0157] Step 5: Synthesis of compound D9-6

[0158] To a 100 mL single-necked flask, add B6-4 (164 mg, 0.22 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (84 mg, 0.22 mmol) and dissolve in N,N-dimethylformamide (3 mL). Diisopropylethylamine (70 mg, 0.54 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. D9-5 (200 mg, 0.18 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 10:1) to afford compound D9-6 (200 mg, 60%).

[0159] LC-MS: 927.5 [M / 2+H] + .

[0160] Step 6: Synthesis of compound D9-7

[0161] Compound D9-6 (200 mg, 0.11 mmol) was added to a 100 mL single-necked flask and dissolved in N,N-dimethylformamide (3 mL). Diethylamine (74 mg, 1.1 mmol) was added to the resulting solution. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was washed three times with petroleum ether and methyl tert-butyl ether by ultrasonication. Filtering afforded compound D9-7 (150 mg, 83.6%).

[0162] LC-MS: 1632.2[M+H] + .

[0163] Step 7: Synthesis of compound D9

[0164] D9-4 (9 mg, 0.018 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.8 mg, 0.018 mmol) were added to a 50 mL single-necked vial and dissolved in N,N-dimethylformamide (0.5 mL). Diisopropylethylamine (5 mg, 0.036 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. D9-7 (25 mg, 0.015 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was filtered and purified by preparative chromatography (column: Boston Prime C18 150*30 mm*5 μm; A: 0.225% FA in Water; B: MeCN) using a gradient separation to obtain D9 (10 mg, 31.4%).

[0165] LC-MS: 1060.1[M / 2+H] + .

[0166] 1H NMR(400MHz, DMSO-d6)δ9.96(s,1H),9.14(d,J=11.7Hz,4H),8.12-7.95(m,3H),7.72–7.48(m,5H),7.39 –7.11(m,8H),5.95(t,J=5.8Hz,1H),5.38(s,2H),5.04(s,2H),4.85-4.56(m,5H),4.55–4.32(m,3H),4. 31–4.11(m,3H),4.08–3.88(m,2H),3.85(s,2H),3.63–3.44(m,26H),3.43-3.40(m,3H),3.26–3.15(m,1 0H),3.14–2.70(m,14H),2.48–2.19(m,4H),2.16-1.87(m,5H),1.86–1.15(m,18H),1.12–0.65(m,34H).

[0167] Example D10: Preparation of Compound D10

[0168] Step 1: Synthesis of 9H-fluoren-9-ylmethyl {[(7S)-7-benzyl-11-{[4-(hydroxymethyl)phenyl]amino}-2,5,8,11-tetrahydroylidene-3,6,9-triazaundec-1-yl]amino}methane

[0169] B20-1 (559.0 mg, 1.0 mmol) and (4-aminophenyl)methanol (123 mg, 1.0 mmol) were added to a 100 mL single-necked bottle. The mixture was dissolved in dichloromethane (5 mL) and methanol (5 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (296.7 mg, 1.2 mmol) was added dropwise to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 93:7) to obtain B20-2 (550.0 mg, 82.8%).

[0170] LC-MS: 664.3[M+H] + .

[0171] Step 2: Synthesis of B20-3

[0172] In a 100 mL single-necked bottle, 9H-fluoren-9-ylmethyl {[(7S)-7-benzyl-11-{[4-(hydroxymethyl)phenyl]amino}-2,5,8,11-tetrahydroylidene-3,6,9-triazaundec-1-yl]amino}methane was added and dissolved in N,N-dimethylformamide (6 mL). Diisopropylethylamine (0.4 mL, 2.55 mmol) and 4-dimethylaminopyridine (0.8 mg, 0.01 mmol) were added to the resulting solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and slurried in methanol to obtain B20-3 (598.0 mg, 72.2%).

[0173] LC-MS:829.3[M+H] + .

[0174] Step 3: Synthesis of compound D10-1

[0175] To a 50 mL single-necked flask, B20-3 (100.0 mg, 0.13 mmol), monomethyl auristatin E (93 mg, 0.13 mmol), and 1-hydroxybenzotriazole (17.6 mg, 0.13 mmol) were added and dissolved in N,N-dimethylformamide (2 mL). The resulting solution was stirred at room temperature for 30 minutes, followed by the addition of diisopropylethylamine (0.06 mL, 0.39 mmol). The reaction mixture was stirred at room temperature for 3 hours, during which time the turbid solution became clear. The reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (dichloromethane:methanol = 12:1) to afford D10-1 (120.0 mg, 65.6%).

[0176] LC-MS: 704.4 [M / 2+H] + .

[0177] Step 4: Synthesis of Compound D10-2 to Compound D10

[0178] Compound D10-2 to compound D10 were synthesized by referring to the synthetic process of D9-6 to D9 to obtain 13 mg of compound D10.

[0179] LC-MS: 1091.0 [M / 2+H] + .

[0180] Example D11: Preparation of Compound D11

[0181] Step 1: Synthesis of compound D11

[0182] The synthesis of this compound was carried out according to the synthesis process of compound D9, except that Val-Cit was replaced by Val-Ala to obtain 15 mg of compound D11.

[0183] LC-MS: 1017.0 [M / 2+H] + .

[0184] Example D12: Preparation of Compound D12

[0185] Step 1: Synthesis of compound D12-4

[0186] The synthesis steps of this compound were based on the synthesis method of Example 223 in patent WO2013175053 A1 to obtain 500 mg of compound D12-4.

[0187] LC-MS: 224.1[M+H] + .

[0188] Step 2: Synthesis of compound D12-7

[0189] D12-6 (350 mg, 1 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (456 mg, 1.2 mmol) were added to a 100 mL single-necked bottle and dissolved in N,N-dimethylformamide (5 mL). Diisopropylethylamine (258 mg, 2 mmol) was added to the resulting solution, and the mixture was stirred at room temperature for 15 minutes. D12-5 (98 mg, 1 mmol) was then added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (15 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (petroleum ether:ethyl acetate = 1:2) to obtain compound D12-7 (330 mg, 76.7%).

[0190] LC-MS: 375.2[M-56+H] + .

[0191] Step 3: Synthesis of compound D12-8

[0192] D12-7 (330 mg, 0.87 mmol) was added to a 100 mL single-necked flask. Glacial acetic acid (4 mL) was added under nitrogen, and after stirring to dissolve, 2,3-dibromomaleic anhydride (222 mg, 0.87 mmol) was slowly added. The mixture was heated to 110°C and stirred overnight under nitrogen. The reaction was monitored by TLC. After cooling the reaction solution to room temperature, the solvent was evaporated to dryness by rotary evaporation. Toluene was then added and evaporated to dryness twice by rotary evaporation to obtain compound D12-8 (500 mg, 93.8%). This was used directly in the next step without purification.

[0193] LC-MS:611.0,613.0,615.0[M+H] + .

[0194] Step 4: Synthesis of compound D12-9

[0195] Compound D12-8 (500 mg, 0.81 mmol) was weighed into a 50 mL round-bottom flask and dissolved in 8 mL of anhydrous dichloromethane under nitrogen. Compound D12-4 (361 mg, 1.62 mmol) was added to the reaction mixture under nitrogen. Once dissolved, DIPEA (209 mg, 1.62 mmol) was slowly added dropwise in an ice bath. Stir for 5 minutes and remove the ice bath. Stir at room temperature under nitrogen for 2 hours. TLC confirmed the reaction was complete. The solvent was evaporated under reduced pressure, and the residue was purified on a C18 column (15% MeCN, 85% 0.1% TFA in Water) using a gradient elution method to afford D12-9 (530 mg, 72.9%).

[0196] LC-MS:897.3[M+H] + .

[0197] Step 5: Synthesis of Compound D12

[0198] To a 50 mL single-necked bottle, add D12-9 (20 mg, 0.022 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8 mg, 0.022 mmol) and dissolve in N,N-dimethylformamide (2 mL). Add diisopropylethylamine (5 mg, 0.036 mmol) to the resulting solution, and stir the mixture at room temperature for 15 minutes. D9-1 (20 mg, 0.018 mmol) is then added to the mixture. The reaction mixture is stirred at room temperature for 3 hours. The reaction mixture is filtered and purified by preparative purification to obtain D12 (13 mg, 36.1%).

[0199] LC-MS: 1002.0 [M / 2+H] + .

[0200] Example D13: Preparation of Compound D13

[0201] The preparation method of this compound was similar to Step 5 of Compound D12, except that Val-Cit was replaced with Val-Ala to obtain 14 mg of Compound D13.

[0202] LC-MS: 958.5 [M / 2+H] + .

[0203] Example D14: Preparation of Compound D14

[0204] The preparation method of this compound was similar to that of compound D12, except that Val-Cit was replaced with Gly-Gly-Phe-Gly to obtain 17 mg of compound D14.

[0205] LC-MS: 1032.5 [M / 2+H] + .

[0206] Example D15: Preparation of Compound D15

[0207] The preparation method of this compound refers to the synthesis method of compound D10, except that Gly-Gly-Phe-Gly is replaced with Ala-Ala-Asn to obtain 10 mg of compound D15.

[0208] LC-MS: 1060.0 [M / 2+H] + .

[0209] Example D16: Preparation of Compound D16

[0210] The preparation method of this compound refers to the synthesis method of compound D14, except that Gly-Gly-Phe-Gly is replaced with Ala-Ala-Asn to obtain 15 mg of compound D16.

[0211] LC-MS: 1001.5 [M / 2+H] + .

[0212] Example D17: Preparation of Compound D17

[0213] The preparation method of this compound was based on the synthesis method of compound D9 to obtain 12 mg of compound D17.

[0214] LC-MS: 1167.6 [M / 2+H] + .

[0215] Example D18: Preparation of Compound D18

[0216] Step 1: Synthesis of 1-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)oxy]tetrahydropyrrole-2,5-dione B25-2

[0217] (2,5,8,11,14,17,20-Heptadioxacosadoca-22-yloxy)acetic acid B25-1 (1 g, 2.5 mmol) was dissolved in anhydrous dichloromethane (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (575 mg, 3 mmol) and N-hydroxysuccinimide (345 mg, 3 mmol) were added to the reaction system, and the reaction was stirred overnight for 3 h. The reaction solution was spin-dried and purified on a silica gel column to obtain B25-2 (1 g, 80.6%).

[0218] LC-MS:496.2[M+H] + .

[0219] Step 2: Synthesis of (2S)-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)-4-[(25-oxyylidene-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)amino]butanoic acid B25-3

[0220] Compound B25-2 (500 mg, 1.0 mmol) and (2S)-4-amino-2-({[(9H-fluoren-9-ylmethyl)oxy]carbonyl}amino)butyric acid (374 mg, 1.1 mmol) were dissolved in N,N-dimethylformamide (8 mL), and triethylamine (202 mg, 2 mmol) was added to the reaction system. The reaction was stirred overnight for 3 h. The reaction solution was spin-dried and purified by liquid phase preparation to obtain B25-3 (550 mg, 76.3%).

[0221] LC-MS:721.5[M+H] + .

[0222] Step 4: Preparation of compound D18

[0223] The preparation method of this compound was based on the synthesis method of compound D9 to obtain 15 mg of compound D18.

[0224] LC-MS: 1046.0 [M / 2+H] + .

[0225] Example E Sequence Expression and Purification of Antibody PF6263507

[0226] Antibody PF6263507 can be prepared according to US20180162937A1, or according to the following method:

[0227] Dilute the cells to 6 × 10 cells using fresh ExpiCHO expression medium (purchased from Thermo Fisher, catalog number: A29133). 6 viable cells / mL, with a viability greater than 95%. Prepare ExpiFectamine CHO reagent: DNA complex at a ratio of 4:1 and incubate at room temperature for 3 minutes, then add the complex to the cells to be transfected and mix well. Incubate at 37°C, 80% humidified air containing 8% CO2. Add ExpiFectamine CHO enhancer and ExpiCHO excipients 18-22 hours after transfection, transfer the culture flask to 32°C, 80% humidified air containing 5% CO2 and continue culturing. After 10 days of cell culture, collect the expression supernatant, centrifuge at high speed to remove cell debris, filter with 0.22um filter membrane, and perform affinity purification using Protein A column (HiTrap MabSelect PrismA protein A column). Rinse the column with PBS to A 280 The reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was changed to PBS for aliquoting. The final purified PF6263507 antibody was subjected to SDS-PAGE and HPLC purity analysis and A 280 Concentration determination.

[0228] Example F ADC conjugate preparation and analysis

[0229] 1. ADC coupling conditions and preparation:

[0230] (1) ADC coupling method 1

[0231] The antibody fraction (PBS 1×) was reduced with a 10 mg / mL TCEP aqueous solution (TCEP = 20 antibody equivalents) at 37°C in 25 mM histidine buffer, pH 6.5, for 2 hours. 10% DMSO organic cosolvent was then added to the system, mixed thoroughly, and then 5 equivalents of the linker-payload DMSO solution were added. The reaction was allowed to proceed on a rotating mixing plate at room temperature for 2 hours. After completion, the solution was ultrafiltration and centrifugation to remove residual small molecules, and the solution was exchanged to 25 mM histidine buffer, pH 5.5, for storage at a concentration of approximately 3 mg / mL for the antibody conjugate product.

[0232] (2) ADC conjugation method 2

[0233] The antibody fraction (PBS 1×) was reduced with a 10 mg / mL TCEP aqueous solution (TCEP = 2-5 times the antibody equivalent) at 37°C in 25 mM histidine buffer, pH 6.5, for 2 hours. 10% DMSO organic cosolvent was then added to the system, mixed thoroughly, and then 5-7 equivalents of vedotin (purchased from MCE, CAS No. 646502-53-6, Cat. No. HY-15575) in DMSO were added. The reaction was allowed to proceed on a rotating mixing plate at room temperature for 2 hours. After completion, the solution was ultrafiltration and centrifugation to remove residual small molecules, and the solution was exchanged to 25 mM histidine buffer, pH 5.5, for storage, with the antibody conjugate product concentration reaching approximately 3 mg / mL.

[0234] The ADC compounds obtained by coupling of the present invention are specifically shown in Table 1.

[0235] Table 1 ADC compounds obtained in the present invention

[0236] 2. ADC analysis method:

[0237] The PB used in the present invention refers to a sodium phosphate buffer solution with disodium hydrogen phosphate and sodium dihydrogen phosphate as the main components. Disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solutions of different pH values ​​are usually prepared using sodium dihydrogen phosphate and disodium hydrogen phosphate solutions of the same concentration.

[0238] ADC purity was determined by SEC and DAR value was determined by hydrophobic chromatography PHIC-UPLC.

[0239] Sample processing: Sample concentration 1.0-5mg / ml, filtered with 0.22um filter membrane

[0240] Common detection methods include:

[0241] Size Exclusion Chromatography SEC-HPLC:

[0242] Sample processing: Sample concentration 1.0-5mg / ml, filtered through 0.22um filter membrane;

[0243] Chromatographic column: TOSOH, TSKgel G3000SWxL, 5 μm, 7.8 mm × 300 mm;

[0244] Mobile phase: 0.2 M PB, 5-15% isopropanol pH 7.0;

[0245] Flow rate: 0.5-1 mL / min;

[0246] Detection wavelength: 280nm & 248nm;

[0247] Column temperature: RT;

[0248] Loading amount: 30 μg;

[0249] SEC chromatography elution method: isocratic elution.

[0250] (2) Hydrophobic interaction chromatography HIC-HPLC

[0251] Chromatographic column: Tosoh, HIC TSK Butyl NPR, 2.5 μm, 4.6 mm × 100 mm

[0252] Column temperature: room temperature

[0253] Mobile phase A: 0.05 M PB, 1.2 M ammonium sulfate, pH 7.0;

[0254] Mobile phase B: 0.05 M PB, pH 7.0, 20% isopropanol;

[0255] Flow rate: 0.5 mL / min;

[0256] Sample load: 30 μg

[0257] Gradient method: increase from 0% to 100% in 20 minutes; detection wavelength: 280nm & 248nm.

[0258] The ADC product coupled by this method was tested for HIC DAR value and SEC purity by HIC-UPLC and SEC-HPLC, respectively. For specific test results, please refer to Table 2-1, Table 2-2 and Figure 1-8.

[0259] Table 2-1 Distribution of different DAR components of ADC1 and ADC3

[0260] Table 2-2 Distribution of different DAR components of ADC2 and ADC4

[0261] (*DAR3 represents an ADC molecule with a drug-to-antibody ratio of 3; DAR4, DAR5, etc. are similar. **"DAR value" represents the average DAR of the ADC product).

[0262] The above results indicate that the ADC1 and ADC3 compounds of the present invention using compound D9 as a payload-linker have significantly excellent uniformity.

[0263] Biological activity experiments

[0264] 1. In vitro efficacy of ADC

[0265] The purpose of the experiment was to detect the in vitro cytotoxic activity of the ADC of the present invention against SKGT-4 (human esophageal cancer tumor), SK-BR-3 (human breast cancer cells), and MDA-MB-468 (human breast cancer cells).

[0266] Experimental equipment:

[0267] 96-well transparent flat-bottom white plate (Corning, #3610)

[0268] RPMI1640 culture medium (Gibco, #A10491-01)

[0269] MEM medium (Gibco, #11095080)

[0270] Fetal Bovine Serum (Sigma-Aldrich, #F8687-500ML)

[0271] Luminescent Cell Viability Assay (Promega, #G7572 / G7571)

[0272] Experimental methods:

[0273] (1) Inhibitory activity of the ADC of the present invention on SKGT-4 cells

[0274] Human esophageal cancer SKGT-4 cells (Nanjing Kebai, Catalog No.: CBP60462) were washed with PBS and digested with 0.25% Trypsin-EDTA for approximately 3-10 minutes. Digestion was terminated with complete cell culture medium. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the cell count was adjusted to 1500 cells / well. The cell plates were incubated in a 37°C, 5% CO2 cell culture incubator overnight.

[0275] In the experiment, a DMSO control group and a test sample group were set up respectively. The cell plate was taken out of the incubator to observe the cell adhesion status. After the cells adhered, 50.00 μL of sample (starting with a final concentration of 100 nM, 5-fold dilution, 9 concentrations) was added to each well, and the plate was gently shaken and placed in the incubator for incubation.

[0276] After 3 days of incubation, 50.0 μL / well CellTiter-Glo TM (Promega, catalog number: G7572 / G7571) working solution, shake on a constant temperature shaker to lyse the cells, and read the plate on a microplate reader after 10 minutes.

[0277] The cell proliferation inhibition rate was calculated as follows: cell proliferation inhibition rate = (1-RLU test sample wells / RLU DMSO control wells) × 100%. The cell proliferation inhibition rate was calculated as follows: cell proliferation inhibition rate = (1-RLU test sample wells / RLU DMSO control wells) × 100%.

[0278] Data analysis: GraphPad Prism 8.0 software was used to plot the sample concentration log value as the horizontal axis and the inhibition percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC of each test sample. 50 The specific results are shown in Table 3 and Figure 9.

[0279] Table 3 Tumor inhibitory activity of ADC of the present invention against SKGT-4

[0280] The experimental results show that ADC1 of the present invention has excellent tumor inhibitory activity.

[0281] (2) Inhibitory activity experiments of the ADC of the present invention on SK-BR-3 and MDA-MB-468 cells

[0282] Human breast cancer cells SK-BR-3 (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Catalog No.: TCHu225) and human breast cancer cells MDA-MB-468 (Nanjing Kebai, Catalog No.: CBP60387) were washed with PBS and then trypsinized with 0.25% Trypsin-EDTA for approximately 3-10 minutes. Digestion was terminated with complete culture medium and the cells were centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded and the cells were resuspended in complete cell culture medium. The cells were counted using a cell counter and the desired cell density was adjusted. 100.0 μL of cell suspension was added to each well (2000 cells / well for SK-BR-3 cells and 1000 cells / well for MDA-MB-468 cells), and the cell plates were incubated overnight in a 37°C, 5% CO2 cell culture incubator.

[0283] A control group and a test group were set up in the experiment. The cell plate was removed from the incubator to observe the cell adhesion status. After the cells attached, 100.0 μL of sample (starting at a final concentration of 200 nM, 5-fold dilution, 8 concentrations) was added to each well. The plate was gently shaken and incubated in a 37°C, 5% CO2 incubator.

[0284] After 6 days of incubation, 100.0 μL of culture supernatant was discarded from each well and 100.0 μL / well of CellTiter-Glo was added. TM (Promega, catalog number: G7572) working solution, shake on a constant temperature shaker to lyse the cells, and read the plate on a microplate reader after 10 minutes.

[0285] The cell proliferation inhibition rate was calculated as follows: cell proliferation inhibition rate % = (1-RLU test sample wells / RLUCell control wells) × 100%.

[0286] Data analysis: GraphPad Prism 8.0 software was used to plot the sample concentration log value as the horizontal axis and the inhibition percentage as the vertical axis. Nonlinear regression (curve fit) analysis was performed on the data to obtain the IC of each test sample. 50 The specific results are shown in Table 4 and Figures 10-11.

[0287] Table 4 Inhibitory activity and target selection specificity of ADC of the present invention against SK-BR-3 and MDA-MB-468

[0288] The experimental results show that the ADCs of the present invention have excellent tumor suppressor activity on cells with high target expression, but ADC3 is much weaker than ADC4 on cells with negative target expression, indicating that the targeting selectivity of ADC3 is significantly stronger than that of ADC4.

[0289] 2. Evaluation of drug efficacy in SKGT-4 tumor-bearing mice

[0290] NPG mice (Beijing Weitongda Biotechnology Co., Ltd., SCXK (Beijing) 2023-0014) were used as test animals to evaluate the efficacy of 5T4-ADC in nude mice bearing human esophageal cancer SKGT-4 xenografts after tail vein injection.

[0291] Under sterile conditions, a suspension of SKGT-4 cells (purchased from ATCC) cultured in vitro was taken and a suspension containing about 5×10 6 SKGT-4 cells were suspended in PBS and mixed with Matrigel (v / v 1:1) and then inoculated subcutaneously into the right axilla of mice (0.2 mL / mouse). The day of inoculation was designated as day 0 (D0). The mice were injected into the tail vein once a week for a total of two doses. Tumor volume and body weight were measured twice a week and the data were recorded. The tumor inhibition rate data on day 28 after administration was TGI (%) = [1-(T 28 -T0) / (V 28 -V0)]×100,T 28 T0 and T1 were the tumor volumes on the 28th and 0th day of drug administration in the experimental groups, respectively. 28 V0 and V1 are the tumor volumes of the blank control group (Vehicle, PBS) on day 28 and day 0, respectively. The tumor inhibition rate data on day 28 after administration are shown in Table 5, and the tumor growth curve on day 28 after administration is shown in Figure 12.

[0292] Table 5 In vivo tumor inhibitory activity of ADC of the present invention in SKGT-4 tumor-bearing mice

[0293] The test results show that the ADC of the present invention has excellent in vivo efficacy.

[0294] 3. Efficacy evaluation in NCI-N87 tumor-bearing mice

[0295] Female BALB / c nude mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were adapted to live in an SPF animal room for 7 days before being used in subsequent studies. NCI-N87 cells (purchased from ATCC) were cultured at a density of 5×10 6 Cells / 0.1 mL (mixed with matrix gel at a ratio of 1:1) were inoculated subcutaneously in the right axilla of nude mice. When the average tumor volume reached 150 mm 3 Around 40 days, the animals with moderate tumor volume were randomly divided into 3 groups: 8 in each group, G1: Vehicle, G2: ADC4 (2 mg / kg), G3: ADC3 (2 mg / kg). On the day of grouping, a single intravenous injection was given, and the vehicle was given PBS. Tumor size was measured and weighed twice a week. The long and short diameters of the tumors were measured using a vernier caliper, and the tumor volume (mm 3 )=0.5×long diameter×short diameter 2 The experiment ended on the 30th day after group administration, and the tumor inhibition rate TGI (%) was calculated as [1-(T 30 -T0) / (V 30 -V0)]×100,T 30 T0 and T1 were the tumor volumes on the 30th and 0th day of drug administration in the experimental groups, respectively. 30 V0 and V1 are the tumor volumes of the blank control group (Vehicle, PBS) on day 30 and day 0, respectively. The tumor inhibition rate data on day 30 after administration are shown in Table 6 and the tumor growth curve on day 30 after administration is shown in Figure 13.

[0296] Table 6 In vivo tumor inhibitory activity of ADC in NCI-N87 tumor-bearing mice

[0297] The test results showed that ADC3 using compound D9 as a linker-load had excellent in vivo efficacy.

[0298] 4. Mouse plasma stability experiment

[0299] All plasma, PBS buffer for dilution, and other reagents used in the experiment were sterilized by filtration using a 0.22 μm microporous filter membrane in a clean bench (or biosafety cabinet) to ensure sterility during the subsequent 21-day incubation process; aseptic operation was required throughout the experiment.

[0300] ADC3 and ADC4 were prepared using mouse plasma as the matrix to a final concentration of 100 μg / mL. Each incubation sample was aliquoted into 0.6 mL EP tubes, totaling 10 tubes per matrix. Each EP tube contained 200 μL of sample, resulting in two samples at each time point. All samples were incubated at 37°C in a 5% CO2 incubator. Samples were collected on Day 0, Day 3, Day 7, Day 14, and Day 21 and immediately frozen at -80°C until testing.

[0301] Take 25 μL of the prepared and incubated plasma sample or biological sample of unknown concentration and add 100 μL of acetonitrile (containing 1 ng / mL MMAE-D8) solution. Vortex mix for 2 minutes. Replace the blank sample with acetonitrile without the internal standard and centrifuge at 4000 rpm (4°C) for 10 minutes. Take 80 μL of the supernatant and add 150 μL of 0.2% formic acid solution. Vortex mix for 2 minutes and centrifuge at 4000 rpm (4°C) for 5 minutes. Transfer the sample to a 96-well plate for LC-MS / MS analysis to quantify the concentration of free MMAE. Specific results are shown in Table 7 and Figure 14.

[0302] Table 7 Plasma stability of ADC in mice

[0303] The experimental results showed that the compound ADC3 using compound D9 as the linker-load had a 21-day load shedding rate in mouse plasma of 2.42%, which was much lower than the 21-day load shedding rate of ADC4 using Vedotin as the load-linker of 13.42%, and had better mouse plasma stability.

[0304] 5. Evaluation of drug efficacy in JIMT-1 tumor-bearing mice

[0305] Female BALB / c nude mice were purchased from Jicui Yaokang and were adapted to live in an SPF animal room for 7 days for subsequent studies. JIMT-1 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured at a density of 5×10 7 Cells / 0.1 mL (mixed with matrix gel at a ratio of 1:1) were inoculated subcutaneously in the right axilla of nude mice. When the average tumor volume reached 150 mm 3At about 14 days, the patients with moderate tumor volume were randomly divided into 5 groups: G1: Vehicle, G2: ADC3 (2 mg / kg), G3: ADC3 (6 mg / kg), G4: ADC4 (2 mg / kg), G5: ADC4 (6 mg / kg), with 6 mice in each group. On the day of grouping, a single intravenous injection was given, and PBS was given to the vehicle. Tumor size was measured and weighed twice a week. The long and short diameters of the tumors were measured with a vernier caliper, and the tumor volume (mm 3 ) = 0.5 × major diameter × minor diameter 2 The experiment ended on the 59th day after group administration, and the tumor inhibition rate TGI (%) was calculated as [1-(T 59 -T0) / (V 59 -V0)]×100,T 59 T0 and T1 were the tumor volumes on the 59th and 0th day of drug administration in the experimental groups, respectively. 59 V0 and V1 are the tumor volumes of the blank control group (Vehicle, PBS) on day 59 and day 0, respectively. The tumor inhibition rate and complete tumor regression rate on day 59 after administration are shown in Table 8, and the tumor growth curve is shown in Figure 15.

[0306] Table 8 In vivo tumor inhibitory activity of ADC of the present invention in JIMT-1 tumor-bearing mice

[0307] The test results showed that in JIMT-1 tumor-bearing mice, ADC3 using compound D9 as a linker-load exhibited significantly better in vivo efficacy than ADC4.

[0308] 6. Pharmacokinetics in rats

[0309] The purpose of the experiment is to detect the pharmacokinetic characteristics of the ADC drug of the present invention in SD rats.

[0310] SD rats (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were randomly divided into groups of 3 males per group and injected with a single sample (the dosing solution was prepared with an appropriate volume of normal saline). Blood samples were collected before administration and 5 minutes, 6 hours, 24 hours, 72 hours, 120 hours, 168 hours, 336 hours, and 504 hours after administration. The concentration of ADC in the plasma samples was determined by LC-MS. The pharmacokinetic parameters of different detection formats were calculated using WinNonlin software. Specific results are shown in Table 9.

[0311] Table 9 Pharmacokinetic properties of the ADC compounds of the present invention in rats

[0312] The test results showed that compound ADC3 using compound D9 as a linker-load had a higher exposure and lower in vivo clearance in rats.

[0313] 7. Rat tolerance test

[0314] Experimental Protocol: Three male rats (SD rats, SPF grade, purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd.) were administered a single tail vein injection of 10 mpk of the drug in each group. Body weight changes, blood routine and biochemical parameters were measured. Clinical observations included body weight and mortality, and abnormal parameters were recorded. Blood routine data for each group on day 4 after administration are shown in Table 10.

[0315] Table 10 Blood routine data of healthy rats and rats in each treatment group at a dose of 10 mpk on the 4th day after administration

[0316] Experimental data showed that at a 10 mpk dose, the ADC4 group using Vedotin as a linker-payload showed significant leukopenia, neutropenia, reticulocyte count, erythrocyte count, and hemoglobin decrease on day 4 after administration. However, the effects of ADC3 using compound D9 as a linker-payload on leukocyte and erythrocyte markers were significantly weaker than those of ADC4. Leukocytes are further divided into lymphocytes, neutrophils, eosinophils, basophils, and monocytes, with lymphocytes and neutrophils being the most important. Leukopenia indicates that the drug has a myelosuppressive effect, which can easily lead to serious secondary infections. Reticulocyte count is an important indicator of bone marrow hematopoietic function. Reticulocyte decrease indicates decreased bone marrow hematopoietic function, which is commonly seen in aplastic anemia and myeloid anemia. These data demonstrate that at 10 mpk, ADC compounds using compound D9 as a linker-payload exhibit lower hematotoxicity.

Claims

1. Antibody drug conjugate represented by formula I or a pharmaceutically acceptable salt thereof: Ab-[-L1-L2-L3-L4-D] q Formula I in, Ab is an antibody portion or antigen-binding fragment; D is a drug molecule, -L1-L2-L3-L4- is a fragment connecting Ab and drug molecule D, and q is selected from 2-9; L1 is a linker portion connected to the antibody, which is selected from the following structures: The end marked with an asterisk * of L1 is connected to Ab; L2 is selected from the following fragments: wherein g, k, and p are each independently selected from 0, 1, 2, 3, 4, and 5; j is selected from 5, 6, 7, 8, and 9, and the end of L2 marked with an asterisk * is connected to L1; L3 is a polypeptide fragment consisting of 2-4 amino acid residues, wherein the amino acid residues are selected from the following α-amino acid residues: glycine, phenylalanine, valine, alanine, asparagine, citrulline, lysine, serine, glutamic acid, and aspartic acid; and the -NH-terminus of L3 is connected to L2; L4 is a chemical bond or L4 is selected from the following groups: -NH-CH2-, Wherein, R3 is selected from or R3 is selected from H, OH, CN, NH2, halogen atoms, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 haloalkoxy; said r is selected from 5, 6, 7, 8, 9; the -NH- end of L4 is connected to L3.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The drug molecule D is selected from camptothecin compounds, auristatin compounds, eribulin compounds, maytansine compounds, calicheamicin compounds, and anthramycin compounds, preferably auristatin compounds, and more preferably MMAE.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: q is selected from 2-6, preferably 3-5, more preferably 3.5-4.5; g and k are each independently selected from 0, 1, 2; p is selected from 1 or 3; j is selected from 6, 7, 8, preferably j is 7; r is selected from 6, 7, 8, preferably r is 7; in one technical solution, j is selected from 7, p is 1 or 3, g is 1, and k is 4.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: R3 is selected from H, F, Cl, methyl, ethyl, preferably R3 is H; or L2 is selected from the following fragments: L3 is selected from a polypeptide fragment consisting of 2-4 of the following amino acid residues: glycine, phenylalanine, valine, alanine, citrulline, asparagine; and the -NH-terminus of L3 is linked to L2, preferably L3 is selected from the following polypeptide fragments: -Val-Cit-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-Asn-; and the -NH-terminus of L3 is linked to L2; L4 is selected from Its -NH-terminus is connected to L3.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: -L1-L2-L3-L4- is selected from the following fragments:

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: -L1-L2-L3-L4-D is selected from the following fragments:

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, characterized in that: Ab is selected from anti-Her2 antibody and anti-5T4 antibody; in one technical solution, the Ab is an anti-Her2 antibody, which comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18; or the Ab is an anti-Her2 antibody, which comprises a heavy chain variable region as shown in SEQ ID NO: 14, and a light chain variable region as shown in SEQ ID NO: 19; or the Ab is an anti-Her2 antibody, which comprises a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 20, or the Ab is trastuzumab; or the Ab is pertuzumab; in another technical solution, the Ab is an antibody 5T4 antibody, and the anti-5T4 antibody has the heavy chain variable region as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4. NO: 3, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; or the anti-5T4 antibody has the heavy chain variable region shown in SEQ ID NO: 4, and the light chain variable region shown in SEQ ID NO: 9; or the anti-5T4 antibody has the heavy chain shown in SEQ ID NO: 5, and the light chain shown in SEQ ID NO: 10; or the anti-5T4 antibody is antibody PF6263507.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, characterized in that: The antibody drug conjugate or a pharmaceutically acceptable salt thereof has the chemical structure described in Formula II: wherein Ab, L3, g, j, p, and q have the same meanings as in any one of claims 1 to 7.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8, characterized in that: L3 can be selected from -Val-Cit-, -Gly-Gly-Phe-Gly-, -Val-Ala-, -Ala-Ala-Asn-; and the -NH-terminus of L3 is connected to -CO-.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8 or 9, characterized in that: j is selected from 6, 7, 8, preferably j is 7; and / or g is selected from 0, 1, 2, preferably g is 1; and / or q is selected from 3-5, preferably 3.5-4.5; and / or p is selected from 1 or 3; in one technical solution, j is 7, g is 1, p is selected from 1 or 3, and q is 3.5-4.5; Ab is an anti-5T4 antibody having HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claims 1-7, characterized in that: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof has a chemical structure shown in Formula III-1 or III-2: wherein Ab, L3, q, and k have the same meanings as in any one of claims 1 to 7.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 11, characterized in that: q is selected from 3-5, preferably 3.5-4.5; k is selected from 0, 1, 2; preferably 1; L3 is selected from -Val-Cit-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Ala-Ala-Asn-, and the -NH-terminus of L3 is connected to the adjacent -CO-.

13. The antibody-drug conjugate of formula II-1 or a pharmaceutically acceptable salt thereof: in, q is 3.5-4.5; Ab has the same meaning as in claim 7 or 10.

14. The following compound or a pharmaceutically acceptable salt thereof:

15. The following structural fragment:

16. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 in the preparation of a drug for treating cancer.

17. Use of the compound or pharmaceutically acceptable salt thereof according to claim 14 or the structural fragment according to claim 15 in the preparation of the antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 13.

18. A pharmaceutical composition comprising the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13.

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