Monoclonal antibody for resisting delta-like ligand 3 and application of monoclonal antibody

Abstract

The invention relates to an anti-delta-like ligand 3 monoclonal antibody and application thereof, and belongs to the technical field of biology. The invention provides a monoclonal antibody for resisting a delta-like ligand 3 (DLL3), and research shows that the monoclonal antibody has high affinity and high specificity to the delta-like ligand 3, and the monoclonal antibody has high endocytosis efficiency in cells with high expression of the delta-like ligand 3; meanwhile, research shows that an antibody-coupled drug obtained by coupling the monoclonal antibody and an anti-tumor drug through a joint has relatively strong target cell killing activity, and the antibody-coupled drug obtained by coupling the monoclonal antibody and the anti-tumor drug through the joint has relatively good in-vivo anti-tumor activity, which can be seen that the antibody-coupled drug has relatively good anti-tumor activity. The monoclonal antibody can effectively target neuroendocrine tumors, and has great application prospects in preparation of neuroendocrine tumor treatment drugs.

Description

Technical Field

[0001] The present invention relates to an anti-delta-like ligand 3 monoclonal antibody and application thereof, belonging to the field of biotechnology. Background Art

[0002] Delta-Like Ligand 3 (DLL3) is a single-pass transmembrane protein attached to the cell surface and a member of the Notch ligand family. DLL3 inhibits the activation of the Notch signaling pathway by interacting with the Notch receptor, contributing to processes such as tumor proliferation, migration, and invasion.

[0003] Delta-like ligand 3 is highly expressed on the cell membrane of neuroendocrine tumors. Therefore, delta-like ligand 3 is a potential drug target for the treatment of neuroendocrine tumors, especially in small cell lung cancer (SCLC). The positive expression of delta-like ligand 3 (≥25%) reaches 85%, and the high expression of delta-like ligand 3 (≥75%) reaches 68%. In addition, its expression is low or not expressed in normal tissues.

[0004] Antibody-drug conjugates (ADCs) are targeted therapeutics that combine the high targeting properties of monoclonal antibodies with the high activity of cytotoxic drugs. In ADCs, monoclonal antibodies are conjugated to biologically active cytotoxic small molecule drugs via cleavable or non-cleavable linkers. These ADCs can deliver cytotoxic drugs directly to tumor cells, minimizing damage to normal cells and thus improving therapeutic efficacy and reducing side effects. The development of anti-DL-ligand-3 monoclonal antibodies with high affinity for DL-ligand-3 is crucial for the development of ADCs targeting neuroendocrine tumors. Summary of the Invention

[0005] To solve the above problems, the present invention provides a monoclonal antibody against delta-like ligand 3 (DLL3), wherein the light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3, and the heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.4, a CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.6.

[0006] In one embodiment of the present invention, the light chain variable region of the monoclonal antibody comprises: (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.20 or SEQ ID NO.32; (b) a derivative polypeptide derived from (a) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (a) and which retains the biological function of the sequence from which it is derived; The heavy chain variable region of the monoclonal antibody comprises: (c) a polypeptide having an amino acid sequence as shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.31; (d) A derivative polypeptide derived from (c) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (c) and retains the biological function of the sequence derived therefrom.

[0007] In one embodiment of the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.20 or SEQ ID NO.32; the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.31.

[0008] In one embodiment of the present invention, the light chain of the monoclonal antibody comprises: (e) a polypeptide having an amino acid sequence as shown in SEQ ID NO. 24, SEQ ID NO. 27 or SEQ ID NO. 30; (f) a derivative polypeptide derived from (a) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (e) and which retains the biological function of the sequence from which it was derived; The heavy chain of the monoclonal antibody comprises: (g) a polypeptide having an amino acid sequence as shown in SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 28 or SEQ ID NO. 29; (h) A derivative polypeptide derived from (c) which has substituted, deleted or added one or more amino acids in the amino acid sequence defined in (g) and retains the biological function of the derived sequence.

[0009] In one embodiment of the present invention, the amino acid sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO.24, SEQ ID NO.27 or SEQ ID NO.30; the amino acid sequence of the heavy chain of the monoclonal antibody is shown as SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.28 or SEQ ID NO.29.

[0010] In one embodiment of the present invention, the light chain and heavy chain of the monoclonal antibody are connected by a disulfide bond.

[0011] In one embodiment of the present invention, the number of substituted, deleted or added amino acids in the derivative polypeptide does not exceed five.

[0012] In one embodiment of the present invention, the number of substituted, deleted or added amino acids in the derivative polypeptide does not exceed three.

[0013] The present invention also provides a multispecific antibody comprising the antigen-binding fragment or single-chain variable region of the above-mentioned monoclonal antibody. Multispecific antibodies are antibodies that can simultaneously bind to two or more different epitopes or antigens.

[0014] The present invention also provides a nucleic acid molecule, which encodes the above-mentioned monoclonal antibody; or, the nucleic acid molecule encodes the above-mentioned multispecific antibody.

[0015] The present invention also provides a recombinant plasmid, which carries the nucleic acid molecule.

[0016] In one embodiment of the present invention, the plasmid vector includes a transient transfection vector integrated with a eukaryotic promoter sequence, a lentiviral plasmid vector integrated with a 5' / 3' terminal repeat sequence and a eukaryotic promoter sequence, or a transposon plasmid vector.

[0017] The present invention also provides a host cell, wherein the host cell is transfected with the above-mentioned recombinant plasmid; or the genome of the host cell is integrated with the above-mentioned nucleic acid molecule.

[0018] In one embodiment of the present invention, the host cells include fungi, bacteria, plant cells and / or animal cells; the animal cells include human embryonic kidney 293 cells (HEK293), a cell line derived from human embryonic kidney 293 cells, Chinese hamster ovary cells (CHO) or a cell line derived from Chinese hamster ovary cells.

[0019] In one embodiment of the present invention, the host cell is prepared by transforming the above-mentioned recombinant plasmid into the host cell.

[0020] The present invention also provides a method for preparing the above-mentioned monoclonal antibody, which comprises: inoculating the above-mentioned host cells into a cell culture medium for culturing to obtain a culture fluid; and separating and extracting the above-mentioned monoclonal antibody from the culture fluid.

[0021] The present invention also provides a drug for treating tumors, wherein the drug contains the above-mentioned monoclonal antibody; or, the drug contains the above-mentioned multispecific antibody.

[0022] In one embodiment of the present invention, the drug is an antibody-drug conjugate; the general structural formula of the antibody-drug conjugate is Ab-[L1-L2-L3-L4-D] p ; Wherein, Ab is the above-mentioned monoclonal antibody; D is a bioactive molecule that has an inhibitory effect on tumor cells; p is the drug loading capacity, and the value of p is an integer or decimal from 1 to 16; L1 is selected from the following structures: 、 、 、 or ; In L1, Represents the connection site, the connection site marked by number 1 is connected to Ab through the S atom, and the connection site marked by number 2 is connected to L2; L2 is ; In L2, Represents the connection site, the connection site marked by number 3 is connected to L1, and the connection site marked by number 4 is connected to L3; Z1 is selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, cycloalkyl, heterocyclic, cyano, amino, nitro or hydroxyl; Z2 is selected from -NR1R2 or -OR3; In Z2, R1 and R2 are each independently selected from -(CH2CH2O) m-CH3, hydrogen atom, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic group, aryl or heteroaryl; or, R1 and R2 together with the connected nitrogen atom form a 4-12 membered heterocyclic group; R3 is selected from hydrogen atom, alkyl, haloalkyl, cycloalkyl, heterocyclic group; In R1, R2 and R3, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; the atoms in the heterocyclic group are each independently selected from C atoms, N atoms, O atoms or S atoms; and the heterocyclic group is substituted by R4; m is an integer from 1 to 24; R4 is selected from -(CH2CH2O) m -CH3, -C(=O)-(CH2CH2O) m -CH3, -NR5-C(=O)-(CH2CH2O) m -CH3, -NR5-C(=O)-CH2-NH-C(=O)-CH2-NH-C(=O)-(CH2CH2O) m -CH3, -C(=O)-CH2-NH-C(=O)-CH2-NH-C(=O)-(CH2CH2O) m -CH3, a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group; In R4, the alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; m is an integer from 1 to 24; R5 is selected from a hydrogen atom, an alkyl group, a cycloalkyl group or a heterocyclic group; In R5, the alkyl, cycloalkyl and heterocyclic groups are each independently substituted by a substituent selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, hydroxyl or hydroxyalkyl; Z3 is selected from -(CH2) i -C(=O)-**、-(CH2) i (OCH2CH2) j -C(=O)-**、-(CH2) i -C(=O)-NH-(CH2CH2O) j -CH2CH2-C(=O)-**, -NH-(CH2) i -C(=O)-**, -NH-(CH2) i (OCH2CH2) j -C(=O)-**or chemical bond; In Z3, i is an integer from 1 to 10; j is an integer from 1 to 20; ** is the 4th position; L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues; In L3, the amino acid residue is a natural amino acid residue or a non-natural amino acid residue; L4 is selected from 、 or chemical bonds; In L4, Represents the connection site. The connection site marked with number 5 is connected to L3, and the connection site marked with number 6 is connected to D.

[0023] In one embodiment of the present invention, D is a cytotoxic compound, immunomodulator, enzyme or kinase inhibitor that has an inhibitory effect on tumor cells.

[0024] In one embodiment of the present invention, D is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Eribulin, exatecan, maytansine or SN-38.

[0025] In one embodiment of the present invention, the value of p is an integer or decimal of 1-3, 3-5, 5-7 or 7-9.

[0026] In one embodiment of the present invention, the L1 is ; In L1, Represents the connection site. The connection site marked with number 1 is connected to Ab through the S atom, and the connection site marked with number 2 is connected to L2.

[0027] In one embodiment of the present invention, L2 is ; In L2, Represents the connection site. The connection site marked with number 3 is connected to L1, and the connection site marked with number 4 is connected to L3.

[0028] In one embodiment of the present invention, Z2 is selected from 、 、 、 、 、 、 、 or ; In Z2, the value of n is an integer from 1 to 13.

[0029] In one embodiment of the present invention, Z3 is selected from -(CH2)i -C(=O)-**or -(CH2) i (OCH2CH2) j -C(=O)-**; in Z3, the value of i is an integer from 1 to 10; the value of j is an integer from 1 to 20; ** is the 4th position.

[0030] In one embodiment of the present invention, the amino acid residues are each independently selected from a phenylalanine residue, an alanine residue, a glycine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue or a valine residue.

[0031] In one embodiment of the present invention, the amino acid residues are each independently substituted with a substituent selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy or cycloalkyl.

[0032] In one embodiment of the present invention, the L3 is selected from a tetrapeptide residue comprising glycine-glycine-phenylalanine-glycine, or a dipeptide residue comprising valine-citrulline or valine-alanine.

[0033] In one embodiment of the present invention, the L1-L2-L3-L4-D is selected from (LP-1), (LP-2), (LP-3), (LP-4), (LP-5), (LP-6), (LP-7), (LP-8), (LP-9), (LP-10), (LP-11), (LP-12), (LP-13), (LP-14), (LP-15), (LP-16), (LP-17), (LP-18) or (LP-19).

[0034] In one embodiment of the present invention, the tumor is a tumor expressing DLL3.

[0035] In one embodiment of the present invention, the tumor expressing DLL3 includes lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), squamous cell carcinoma (e.g., esophageal squamous cell carcinoma, cervical cancer), brain cancer (e.g., glioma, neuroblastoma), thyroid cancer (e.g., medullary thyroid carcinoma), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), hepatobiliary cancer (e.g., liver cancer, bile duct cancer, gallbladder cancer), pancreatic cancer, gastrointestinal cancer (e.g., gastric cancer, colorectal cancer), kidney cancer, ovarian cancer, endometrial cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer and / or melanoma.

[0036] The present invention also provides use of the above monoclonal antibody, the above multispecific antibody, the above nucleic acid molecule, the above recombinant plasmid, the above host cell, or the above method in preparing a drug for treating tumors.

[0037] In one embodiment of the present invention, the tumor is a tumor expressing DLL3.

[0038] In one embodiment of the present invention, the tumor expressing DLL3 includes lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), squamous cell carcinoma (e.g., esophageal squamous cell carcinoma, cervical cancer), brain cancer (e.g., glioma, neuroblastoma), thyroid cancer (e.g., medullary thyroid carcinoma), malignant pleural mesothelioma, breast cancer (e.g., triple-negative breast cancer), hepatobiliary cancer (e.g., liver cancer, bile duct cancer, gallbladder cancer), pancreatic cancer, gastrointestinal cancer (e.g., gastric cancer, colorectal cancer), kidney cancer, ovarian cancer, endometrial cancer, bladder cancer, prostate cancer, testicular cancer, adrenal cancer and / or melanoma.

[0039] The technical solution of the present invention has the following advantages: 1. The present invention provides a monoclonal antibody against delta-like ligand 3 (DLL3). The light chain variable region of the monoclonal antibody comprises a CDR1 with an amino acid sequence as set forth in SEQ ID NO.1, a CDR2 with an amino acid sequence as set forth in SEQ ID NO.2, and a CDR3 with an amino acid sequence as set forth in SEQ ID NO.3; the heavy chain variable region comprises a CDR1 with an amino acid sequence as set forth in SEQ ID NO.4, a CDR2 with an amino acid sequence as set forth in SEQ ID NO.5, and a CDR3 with an amino acid sequence as set forth in SEQ ID NO.6. Experiments have demonstrated that the monoclonal antibody has high affinity and specificity for DLL3 and exhibits high endocytosis efficiency in cells with high DLL3 expression. Furthermore, studies have demonstrated that the antibody-drug conjugate obtained by conjugating the monoclonal antibody to an anti-tumor drug via a linker exhibits strong target cell cytotoxicity and exhibits excellent in vivo anti-tumor activity. It can be seen that the monoclonal antibody can effectively target neuroendocrine tumors with high expression of δ-like ligand 3, and has great application prospects in the preparation of therapeutic drugs for neuroendocrine tumors with high expression of δ-like ligand 3.

[0040] 2. The present invention provides a drug for treating tumors, comprising a monoclonal antibody against delta-like ligand 3. The light chain variable region of the monoclonal antibody comprises a CDR1 with an amino acid sequence as set forth in SEQ ID NO.1, a CDR2 with an amino acid sequence as set forth in SEQ ID NO.2, and a CDR3 with an amino acid sequence as set forth in SEQ ID NO.3; and a heavy chain variable region comprises a CDR1 with an amino acid sequence as set forth in SEQ ID NO.4, a CDR2 with an amino acid sequence as set forth in SEQ ID NO.5, and a CDR3 with an amino acid sequence as set forth in SEQ ID NO.6. Experiments have shown that the monoclonal antibody contained in the drug has high affinity and specificity for delta-like ligand 3 and exhibits high endocytosis efficiency in cells with high delta-like ligand 3 expression. Furthermore, experiments have demonstrated that the drug can significantly reduce tumor volume in NCI-H82 and SHP-77 tumor-bearing mice. It can be seen that the drug can effectively target neuroendocrine tumors with high expression of δ-like ligand 3 and has good in vivo anti-tumor activity, and has great application prospects in the treatment of neuroendocrine tumors with high expression of δ-like ligand 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : The endocytosis results of different antibodies in NCI-H82 cells.

[0042] Figure 2 : The endocytosis results of different antibodies in SHP-77 cells.

[0043] Figure 3 : Specificity detection results of JFab13b antibody.

[0044] Figure 4 : The synthetic route of compound LP-1.

[0045] Figure 5 : Mass spectrometry results of compound LP-1.

[0046] Figure 6 : The synthetic route of compound LP-2.

[0047] Figure 7 : Mass spectrometry results of compound LP-2.

[0048] Figure 8 : The synthetic route of compound LP-3.

[0049] Figure 9 : Mass spectrometry results of compound LP-3.

[0050] Figure 10 : The synthetic route of compound LP-4.

[0051] Figure 11 : Mass spectrometry results of compound LP-4.

[0052] Figure 12 : The synthetic route of compound LP-5.

[0053] Figure 13 : Mass spectrometry results of compound LP-5.

[0054] Figure 14 : The synthetic route of compound LP-6.

[0055] Figure 15 : Mass spectrometry results of compound LP-6.

[0056] Figure 16 : The synthetic route of compound LP-7.

[0057] Figure 17 : Mass spectrometry results of compound LP-7.

[0058] Figure 18 : The synthetic route of compound LP-8.

[0059] Figure 19 : Mass spectrometry results of compound LP-8.

[0060] Figure 20 : The synthetic route of compound LP-9.

[0061] Figure 21: Mass spectrometry results of compound LP-9.

[0062] Figure 22 : The synthetic route of compound LP-10.

[0063] Figure 23 : Mass spectrometry results of compound LP-10.

[0064] Figure 24 : The synthetic route of compound LP-11.

[0065] Figure 25 : Mass spectrometry results of compound LP-11.

[0066] Figure 26 : The synthetic route of compound LP-12.

[0067] Figure 27 : Mass spectrometry results of compound LP-12.

[0068] Figure 28 : The synthetic route of compound LP-13.

[0069] Figure 29 : Mass spectrometry results of compound LP-13.

[0070] Figure 30 : The synthetic route of compound LP-14.

[0071] Figure 31 : Mass spectrometry results of compound LP-14.

[0072] Figure 32 : The synthetic route of compound LP-15.

[0073] Figure 33 : Mass spectrometry results of compound LP-15.

[0074] Figure 34 : The synthetic route of compound LP-16.

[0075] Figure 35 : Mass spectrometry results of compound LP-16.

[0076] Figure 36 : The synthetic route of compound LP-17.

[0077] Figure 37 : Mass spectrometry results of compound LP-17.

[0078] Figure 38 : The synthetic route of compound LP-18.

[0079] Figure 39 : Mass spectrometry results of compound LP-18.

[0080] Figure 40 : The synthetic route of compound LP-19.

[0081] Figure 41 : Mass spectrometry results of compound LP-19.

[0082] Figure 42 :The anti-tumor activity of ADC was detected in SHP-77 transplanted tumor model.

[0083] Figure 43 : Body weight changes in SHP-77 transplanted tumor model mice.

[0084] Figure 44 :The anti-tumor activity of ADC was detected in the NCI-H82 xenograft tumor model.

[0085] Figure 45 : Body weight changes in mice with NCI-H82 transplanted tumor model. DETAILED DESCRIPTION

[0086] In order to make the present invention easier to understand, certain technical terms are specifically defined below. Unless otherwise explicitly stated elsewhere in this document, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0087] Antibody: refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only intact antibody molecules but also fragments of such antibodies and variants (including derivatives) of such antibodies and antibody fragments.

[0088] Antibody heavy chain: consists of a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains: CH1, CH2, and CH3.

[0089] Antibody light chain: comprises a light chain variable region (abbreviated herein as "VL") and a light chain constant region containing one domain, CL.

[0090] FR (Framework Region): The term framework region refers to four conserved polypeptide segments within the light chain variable domain and the heavy chain variable domain.

[0091] CDR (Complementarity Determining Region): The term "complementarity determining region" or "CDR" refers to the regions within the light and heavy chain variable domains that primarily contribute to antigen binding. The light and heavy chain variable domains each contain three CDR regions: LCDR1, LCDR2, and LCDR3; and HCDR1, HCDR2, and HCDR3. Antibodies of the present invention include, but are not limited to, monoclonal, multispecific, human, or chimeric antibodies, single-chain antibodies, scFv fragments, and Fab fragments. Antibody molecules of the present invention can be of any immunoglobulin type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, gA1, and IgA2), or subclass.

[0092] Constant region: The constant region of an antibody is located near the carbon-terminus of the H chain and the L chain. The heavy chain constant region is divided into CH1, CH2, and CH3, while the light chain constant region is CL. The CH3 region is involved in binding to cell membrane receptors, while CH2 is involved in the complement activation pathway and serves as the complement binding site.

[0093] Antibody acquisition: The antibodies of the present invention can be prepared using techniques well known in the art, such as the hybridoma method, recombinant DNA technology, phage display technology, synthetic technology, or a combination of these technologies, or other techniques known in the art.

[0094] Antibody discovery: The monoclonal antibody of the present invention obtained by hybridoma method is preferably a mouse anti-human DLL3 monoclonal antibody.

[0095] Antibody humanization: The mouse anti-human DLL3 monoclonal antibody of the present invention is a monoclonal antibody with human FR regions formed by humanization engineering.

[0096] Antibody drug conjugate (ADC): A conjugate obtained by connecting an antibody (or its antigen-binding fragment) to a drug directly or through a linker.

[0097] Linker: A chemical structure or bond that is attached to an antibody at one end and to a drug at the other. Other linkers can also be attached to the antibody or drug. Linker attachment to antibodies can be accomplished in a variety of ways, such as via surface lysines, reductive coupling to oxidized carbohydrates, cysteine residues released by reduction of interchain disulfide bonds, reactive cysteine residues engineered at specific sites, and tags containing the acyl donor glutamine or endogenous glutamine made reactive by engineering the polypeptide in the presence of transglutaminase and an amine.

[0098] Drug loading, also known as the drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated per antibody in an ADC. This can range, for example, from about 1 to about 10 drugs conjugated per antibody, and in certain embodiments, from about 1 to about 8 drugs conjugated per antibody. Drug loading can be expressed as p, which can be a decimal or integer. Drug loading can be measured using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, HIC, and RP-HPLC.

[0099] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0100] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0101] The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance (HNMR) and / or mass spectrometry (MS). HNMR was performed using a Bruker 400 MHz NMR instrument, with deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (d6-DMSO) as the solvent, and tetramethylsilane (TMS) as the internal standard. All chemical shift values (δ) are given in parts per million (ppm). Mass spectrometry (MS) was performed using a Thermo Fisher Vanquish & TSQ Quantis™ instrument. Preparative liquid chromatography was performed using a Gilson GX-281 instrument and a Welch Xtimate C18 30×150 mm, 5 μm column. Reactions were monitored by thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS), using developing systems including, but not limited to, dichloromethane and methanol and petroleum ether and ethyl acetate. The volume ratio of the solvents was adjusted according to the polarity of the compound, including, but not limited to, the addition of triethylamine or acetic acid.

[0102] Experimental Example 1: Preparation, Extraction and Identification of Monoclonal Antibodies Against Delta-like Ligand 3 (DLL3) 1. Mouse immunization Five BALB / c mice and five C57bl / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.) were immunized with human DLL3 target protein (purchased from Biopsy). The specific immunization procedure was as follows: The mice were given the first immunization, the first booster immunization, the second booster immunization, and the third booster immunization on day 0, day 14, day 28, and day 56, respectively. During the immunization period, blood was collected from the mice 4 days before the first immunization, on day 21, and on day 35, and the serum was separated. The antibody titer in the mouse serum was detected by ELISA. Two mice with high antibody titers in the serum were selected for booster immunization to obtain δ-like ligand 3-immunized mice. During the first immunization, the human DLL3 target protein was first diluted with physiological saline to obtain a dilution (dilution ratio of 1 mg:1 mL); the dilution was then mixed with Freund's complete adjuvant (purchased from Shanghai Yisheng) at a volume ratio of 1:1 to obtain an immune preparation; the immune preparation was then subcutaneously injected into the mice at a dose of 50 μg protein per mouse to complete the first immunization; after the first immunization, Freund's incomplete adjuvant (purchased from Shanghai Yisheng) was used for subsequent booster immunizations, among which the first and third booster immunizations were intraperitoneal injections, and the second booster immunization was subcutaneous injection. The injection dose of the three booster immunizations was 25 μg protein per mouse.

[0103] 2. Spleen cell fusion Two mice with high neutralizing antibody titers were selected based on serum titers. The spleens were dissected and ground into single splenocytes. The single splenocytes were washed once with serum-free DMEM medium (purchased from Gibco) and centrifuged at 300 g for 8 minutes. The supernatant was discarded and the spleen cell pellet was collected. The spleen cell pellet was resuspended in serum-free DMEM medium and the number of spleen cells was counted to obtain a spleen cell suspension. Mouse plasma cells were fused with sp20 cells (purchased from ATCC) to obtain fused cells (using the Edward A. Greenfield electrofusion protocol, with a fusion efficiency of approximately one hybridoma per 200 B cells; for details, see the reference "Schmitt, JJ et al. "Efficient generation of stable antibody forming hybridoma cells by electrofusion."). Hybridoma vol. 8,1 (1989):107-15."); The fused cells were seeded into 384-well plates and cultured and screened using HAT complete medium (purchased from Gibco).

[0104] 3. Monoclonal Antibody Screening The cell culture supernatant obtained by culture and screening was collected, and monoclonal antibody screening was performed by ELISA and flow cytometry to obtain the high-affinity antibody mJFab8.

[0105] The ELISA detection process is as follows: antigen A (human DLL3 protein, purchased from Biopsies, the complete amino acid sequence of human DLL3 protein is shown in SEQ ID NO. 16), antigen B (cynomolgus macaque DLL3 protein, purchased from Biopsies, the complete amino acid sequence of cynomolgus macaque DLL3 is shown in SEQ ID NO. 17) or antigen C (mouse DLL3 protein, purchased from Biopsies, the complete amino acid sequence of mouse DLL3 is shown in SEQ ID NO. 18) is coated with a coating buffer (Nanjing GenScript Co., Ltd.). NO.43) were diluted to a concentration of 0.5 μg / mL to obtain antigen dilution solutions; the antigen dilution solutions were added to the ELISA plate at a volume of 100 μL / well, incubated at 4°C overnight (16 h), and then the supernatant in the wells was discarded to obtain antigen-coated ELISA plates; blocking buffer (Nanjing GenScript Company) was added to the ELISA plate coated with the primary antibody at a volume of 100 μL / well, incubated at 37°C for 1 h, and then the supernatant in the wells was discarded to obtain blocked ELISA plates; human lovatuzumab ADC (purchased from Nanjing Pengbo Bio) and mouse lovatuzumab ADC (purchased from Nanjing Pengbo Bio) were used as positive controls, and human IgG (purchased from Nanjing Pengbo Bio) was used as a negative control. The antibody to be tested (i.e., the cell culture supernatant obtained by culture screening) was quantified and then serially diluted to obtain primary antibody dilution solutions of different concentrations; Different concentrations of primary antibody dilutions were added to the blocked ELISA plate at a volume of 100 μL / well, incubated at 37°C for 1 hour, and then the supernatant in the wells was discarded to obtain the ELISA plate incubated with the primary antibody; HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-human IgG (purchased from Jackson) was diluted to the recommended working concentration using blocking buffer according to the instructions of the secondary antibody to obtain the secondary antibody dilution; the secondary antibody dilution was added to the antibody-incubated ELISA plate at a volume of 100 μL / well, incubated at 37°C for 330 minutes, and then the supernatant in the wells was discarded to obtain the ELISA plate incubated with the secondary antibody; the absorbance of each well in the ELISA plate incubated with the secondary antibody at 450 nm was detected using a multifunctional microplate reader (purchased from Beijing Kaiao Technology Co., Ltd.); according to the measured absorbance, GraphPad Prism was used to fit the EC50 values of the test antibodies, which were used to measure their affinity for delta-like ligand 3. For ELISA assays, all test antibodies were initially diluted to a 10 mM concentration, followed by a three-fold serial dilution. The ELISA results are shown in Tables 1 and 2.

[0106] The flow cytometric assay process involved testing each monoclonal antibody separately using CHO-K1 (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) as the negative cell line and a CHO-K1 / DLL3-overexpressing cell line (purchased from Nanjing Pengbo Biotechnology Co., Ltd.) as the positive cell line. Human and mouse lovatuzumab ADCs served as positive controls, human IgG served as the isotype control, and PBS buffer served as the negative control. The assay process involved incubating the monoclonal antibody samples with each of the two cell lines at 4°C, washing, incubating with the secondary antibody, and then washing and analyzing the flow cytometer. (For detailed flow cytometric assay protocols, see the reference "Tabatabaei, Mahdis Sadat, and Marya Ahmed. "Enzyme-Linked Immunosorbent Assay (ELISA).") Methods in molecular biology (Clifton, NJ) vol. 2508 (2022): 115-134.”). Alexa Fluor® 647 AffiniPure goat anti-human IgG, Fcγ fragment-specific antibody (Jackson, 109-605-098, 144613) was used as the secondary antibody for the tested monoclonal antibodies and the human lovatuzumab ADC; Alexa Fluor® 647 AffiniPure goat anti-mouse IgG, Fcγ fragment-specific antibody (Nanjing Pengbo Biotechnology) was used as the secondary antibody for the mouse lovatuzumab ADC. Flow cytometry results are shown in Table 3.

[0107] As shown in Tables 1 and 2, a total of 10 hybridoma cell lines producing mouse-derived monoclonal antibodies against delta-like ligand 3 were screened by ELISA. The 10 hybridoma cell lines and the mouse-derived monoclonal antibodies against delta-like ligand 3 secreted by them were named mJFab1, mJFab2, mJFab3, mJFab4, mJFab5, mJFab6, mJFab7, mJFab8, mJFab9, and mJFab10, respectively. Among these monoclonal antibodies, except for mJFab5 which has a poor affinity, the other antibody samples have high affinity for delta-like ligand 3.

[0108] As shown in Table 3 , among the monoclonal antibodies mJFab1, mJFab2, mJFab3, mJFab4, mJFab5, mJFab6, mJFab7, mJFab8, mJFab9, and mJFab10, mJFab5 bound weakly to CHO-K1 cells with high expression of δ-like ligands, while the remaining antibody samples could bind to CHO-K1 / human DLL3.

[0109] Table 1 ELISA test results

[0110] Table 2 ELISA test results

[0111] Table 3 Flow cytometry results

[0112] 4. Antibody sequence determination The hybridoma cell line mJFab8 obtained by screening was expanded and cultured, and total RNA was extracted using Trizol (purchased from ThermoFisher). Amplification was performed using mouse-Ig degenerate primers (purchased from Nanjing GenScript), and finally, sequencing was performed to obtain the CDR sequences of the antibody variable regions of the monoclonal antibody mJFab8. The sequences are shown in Table 4. The amino acid sequence of the light chain variable region of the monoclonal antibody mJFab8 is shown in SEQ ID NO. 32, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 31.

[0113] Table 4 Antibody variable region CDR sequences

[0114] 5. Antibody humanization The monoclonal antibody mJFab8 was humanized using the bioMOE plug-in. The CDR transplantation parameter was selected, that is, the CDR residues were fixed unchanged. The annotation method was selected as kabbat. Five humanized light chains and seven humanized heavy chains were generated. The variable region sequences of the humanized light chains and humanized heavy chains are shown in Table 5.

[0115] The five humanized light chains and seven humanized heavy chains were spliced with the human IgG1 heavy chain constant region (amino acid sequence shown in SEQ ID NO. 21) and the human IgG kappa chain constant region (amino acid sequence shown in SEQ ID NO. 22), respectively, to form complete humanized antibody light and heavy chain sequences, thereby obtaining five humanized antibodies, namely, humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b. The amino acid sequence of the light chain of humanized antibody JFab4 is shown in SEQ ID NO. 24 (containing the VL-hum5 fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 23 (containing the VH-hum6 fragment); the amino acid sequence of the light chain of humanized antibody JFab12 is shown in SEQ ID NO. 27 (containing the VL-hum2 fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 25 (containing the VH-hum13 fragment); the amino acid sequence of the light chain of humanized antibody JFab13 is shown in SEQ ID NO. The amino acid sequence of the light chain of the humanized antibody JFab12b is shown in SEQ ID NO. 30 (containing the JFab12b / VL fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 28 (containing the VH-hum16 fragment); the amino acid sequence of the light chain of the humanized antibody JFab13b is shown in SEQ ID NO. 30 (containing the JFab13b / VL fragment), and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 29 (containing the JFab13b / VH fragment).

[0116] Table 5 Humanized sequences

[0117] 6. Humanized antibody screening The humanized antibody coding sequence was derived, synthesized, and cloned into the PTT5 antibody expression vector (performed by Anhui General Biotechnology Co., Ltd.). The antibody light and heavy chains were expressed and purified (for details on expression and purification methods, see the reference "Kunert, Renate, and David Reinhart. "Advances in recombinant antibody manufacturing."). Applied microbiology and biotechnologyvol. 100,8 (2016): 3451-61.”), humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b were obtained. Affinity measurements of the humanized antibodies JFab4, JFab12, JFab13, JFab12b, and JFab13b were performed using Biacore T200 (Cytiva) using the monoclonal antibody mJFab8 as a control. The affinity measurement results are shown in Tables 6-7. (In addition to JFab4, JFab12, JFab13, JFab12b, and JFab13b, Tables 6-7 also show other humanized antibodies obtained from the same batch as JFab4, JFab12, JFab13, JFab12b, and JFab13b as controls.)

[0118] As shown in Tables 6 and 7, the equilibrium dissociation constant between the humanized antibody JFab13b and the DLL3 antigen is the lowest. This indicates that JFab13b has the best affinity for the DLL3 target among all the humanized antibodies.

[0119] Table 6 Affinity determination results

[0120] Table 7 Affinity determination results

[0121] 7. Expression and purification of humanized antibodies The humanized antibody coding sequence was derived for synthesis and cloned into the PTT5 antibody expression vector (completed by Anhui General Biotechnology Co., Ltd.), obtaining a recombinant vector expressing the antibody heavy chain and a recombinant vector expressing the antibody light chain. EXPI293F cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were subcultured using serum-free cell culture medium (purchased from Aupromycin) and the cell density was adjusted to 3×10 6After 100 cells / mL, recombinant vectors expressing the antibody heavy chain and recombinant vectors expressing the antibody light chain were transiently transfected into EXPI293F cells using a transfection reagent (purchased from Optum). After transfection, cell density and viability were determined by trypan blue cell counting. Six days after transfection, the cell culture supernatant was collected and affinity purified. The purified protein sample was analyzed by SEC HPLC. The SEC HPLC analysis results are shown in Table 8. Among them, the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab4 is shown in SEQ ID NO.34, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab4 is shown in SEQ ID NO.33; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab12 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab12 is shown in SEQ ID NO.35; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab13 is shown in SEQ ID NO.36, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab13 is shown in SEQ ID NO.37; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab12b is shown in SEQ ID NO.40, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab12b is shown in SEQ ID NO.38; the nucleotide sequence of the gene encoding the light chain variable region of the humanized antibody JFab13b is shown in SEQ ID NO. The nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab13b is shown in SEQ ID NO.40, and the nucleotide sequence of the gene encoding the heavy chain variable region of the humanized antibody JFab13b is shown in SEQ ID NO.39.

[0122] Table 8 SEC HPLC test results

[0123] 8. Humanized Antibody Internalization Detection The endocytic activity of the antibodies was detected using human small cell lung cancer SHP-77 (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences) and human small cell lung cancer NCI-H82 (purchased from the Shanghai Cell Bank, Chinese Academy of Sciences). The endocytic activity assay was performed as follows: the antibody to be tested was diluted to a concentration of 15 μg / mL in PBS buffer containing 2% (v / v) fetal bovine serum (FBS) to obtain the antibody dilution solution; for each antibody, 4 aliquots of 1.5×10 5After the cells were plated in a 96-well U-shaped plate, the supernatant was discarded by centrifugation. The antibody dilution to be tested was added to the 96-well U-shaped plate at a volume of 200 μL / well and incubated on ice for 45 min. After the incubation, the cells were centrifuged at 4°C and the supernatant was discarded. 150 μL / well of PBS buffer containing 2% (v / v) FBS (fetal bovine serum) was added to the 96-well U-shaped plate and the supernatant was discarded by centrifugation. This operation was repeated twice. After the cells in the 96-well U-shaped plate were resuspended with 200 μL / well PBS buffer, three aliquots of cells were taken out and incubated in a 37°C cell culture incubator for 4 h, 1 h, and 0 h respectively. The remaining aliquot of cells was placed on ice for 4 h. After the incubation time was completed, all sample cells were placed at 4°C and centrifuged and the supernatant was discarded. Alexa Fluor 647-labeled goat anti-human IgG (Jackson, Cat. No. 109-605-098, 2% FBS-PBS) was added. 1:800 dilution) was added to a 96-well U-shaped plate at a volume of 100 μL / well and incubated on ice for 30 minutes. After the incubation, the cells were centrifuged at 4°C and the supernatant was discarded. 150 μL / well of PBS buffer containing 2% (v / v) FBS (fetal bovine serum) was added to a 96-well U-shaped plate and the supernatant was discarded. The operation was repeated twice. The cells in the 96-well U-shaped plate were resuspended with 200 μL / well of PBS buffer containing 2% (v / v) FBS (fetal bovine serum) and the cells were incubated with Attune ® The mean fluorescence intensity in the wells was detected by NxT (purchased from Thermo). The internalization rate of different test antibodies in SHP-77 cells or NCI-H82 cells was calculated according to the formula: internalization rate (%) = 100-(MFI of the sample incubated at 37°C at that time point / MFI of the control sample incubated at 4°C at that time point) × 100. The internalization effect of the test antibodies in small cell lung cancer cells with high expression of delta-like ligand 3 was then evaluated. The fluorescence value statistics are shown in Tables 9 and 10, and the internalization rate (%) is shown in Figure 1~Figure 2 .

[0124] The cell surface antibody loading can be expressed by the mean fluorescence intensity (MFI), and the fluorescence intensity decreases with the extension of incubation time, which indicates that the antibody is internalized by the cell, the cell surface antibody loading decreases, and the fluorescence intensity decreases. Figure 1~Figure 2 It can be seen that the humanized antibodies JFab12, JFab13, JFab12b and JFab13b can all be internalized by small cell lung cancer cells with high expression of delta-like ligand 3 (DLL3) and the internalization efficiency is high.

[0125] Table 9 Fluorescence results of antibody internalization detected by flow cytometry (NCI-H82)

[0126] Table 10 Fluorescence results of antibody internalization detected by flow cytometry (SHP-77)

[0127] 9. Study on the specificity of humanized antibodies Immunoblotting was performed using the JFab13b antibody with human DLL1 recombinant protein (purchased from Yisheng Biotechnology, with the complete amino acid sequence shown in SEQ ID NO.41), human DLL3 recombinant protein (purchased from Biopsies, with the complete amino acid sequence shown in SEQ ID NO.16), or human DLL4 recombinant protein (purchased from Huamei Biotechnology, with the complete amino acid sequence shown in SEQ ID NO.42) to determine the specificity of the antibody (for details of the immunoblotting assay, see the reference “Hirano, Seishiro. “Western blot analysis.”). Methods in molecular biology (Clifton, NJ) vol. 926 (2012):87-97.”). In immunoblotting experiments, all antigens contained a histidine His tag and could also be detected by immunoblotting using anti-His tag antibodies. JFab13b and anti-His tag antibodies (purchased from Mitaka Bio) were used as primary antibodies and were conjugated with rabbit anti-human IgG-HRP (purchased from Solebro) and rabbit anti-mouse IgG-HRP (purchased from Solebro) for color development, respectively. The color development results are shown in Figure 3 .

[0128] Depend on Figure 3 It can be seen that JFab13b only binds to human DLL3 recombinant protein, and does not bind to DLL1 recombinant protein and DLL4 recombinant protein, proving that JFab13b specifically binds to DLL3 recombinant protein.

[0129] Example 1-1: A monoclonal antibody against delta-like ligand 3 This example provides an anti-δ-like ligand 3 monoclonal antibody JFab4, the amino acid sequence of the light chain of the monoclonal antibody JFab4 is shown in SEQ ID NO.24, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.23.

[0130] Example 1-2: A monoclonal antibody against delta-like ligand 3 This example provides an anti-δ-like ligand 3 monoclonal antibody JFab12, the amino acid sequence of the light chain of the monoclonal antibody JFab12 is shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.25.

[0131] Example 1-3: A monoclonal antibody against delta-like ligand 3 This example provides an anti-δ-like ligand 3 monoclonal antibody JFab13, the amino acid sequence of the light chain of the monoclonal antibody JFab13 is shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.26.

[0132] Example 1-4: A monoclonal antibody against delta-like ligand 3 This example provides an anti-δ-like ligand 3 monoclonal antibody JFab12b, the amino acid sequence of the light chain of the monoclonal antibody JFab12b is shown in SEQ ID NO.30, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.28.

[0133] Example 1-5: A monoclonal antibody against delta-like ligand 3 This example provides an anti-δ-like ligand 3 monoclonal antibody JFab13b, the amino acid sequence of the light chain of the monoclonal antibody JFab13b is shown in SEQ ID NO.30, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.29.

[0134] Example 2-1: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-1, wherein the linker-cytotoxin LP-1 has the following structure: .

[0135] The preparation method of the linker-cytotoxin LP-1 comprises the following steps: Step 1: Dissolve 2-bromo-5-nitrophenol (0.94 g, 1.0 eq, CAS No. 52427-05-1) in N,N-dimethylformamide (40 mL) to obtain a solution. 1-Boc-4-methanesulfonyloxypiperidine (1.80 g, 1.5 eq, CAS No. 141699-59-4) and potassium carbonate (1.20 g, 2.0 eq) were added to the solution in sequence. The mixture was stirred until uniformly mixed and then stirred at 90°C for 3 hours to obtain a reaction solution. After thin-layer chromatography (TLC) indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL x 3). The organic phase was collected and washed with saturated brine (40 mL x 1) and then concentrated under reduced pressure to obtain compound 1-1. Compound 1-1 was used directly in the synthesis of compound 1-2 without purification. Compound 1-1 was a yellow oil (1.70 g, yield 98%). The mass spectrum data of compound 1-1 was: ESI-MS (m / z): 401.08 [M+H] + .

[0136] Step 2: Compound 1-1 (1.70 g, 1.00 eq) was dissolved in N,N-dimethylformamide (10 mL) to obtain a solution; benzyl acrylate (1.37 g, 2.0 eq, CAS No.: 2495-35-4), palladium acetate (25 mg, 0.025 eq), triphenylphosphine (0.11 g, 0.1 eq) and sodium bicarbonate (0.54 g, 1.5 eq) were added to the solution in sequence, and the mixture was stirred and mixed, and then stirred at 100 ° C for 4 hours under a nitrogen atmosphere to obtain a reaction mixture. After thin-layer chromatography indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (20 mL x 4). The organic phase was washed with water (40 mL x 2) and then with saturated brine (40 mL x 1), then concentrated under reduced pressure and purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 1-2. Compound 1-2 was obtained as a yellow oil (1.86 g, yield 89%). The mass spectrometric data of compound 1-2 were: ESI-MS (m / z): 483.23 [M+H] + .

[0137] Step 3: Compound 1-2 (1.71 g, 1.00 eq) was dissolved in ethyl acetate (15 mL) to obtain a solution; after cooling the solution to 0°C, a hydrogen chloride ethyl acetate solution (4 mol / L, 13.5 mL, 15 eq) was added to the solution, and the mixture was stirred at 0°C for 24 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first concentrated under reduced pressure, then the pH was adjusted to 9 with a saturated sodium carbonate aqueous solution, and then extracted with ethyl acetate (20 ml × 4), and the organic phase was collected; the organic phase was first washed with water (40 ml), then with saturated brine (40 ml), and then concentrated under reduced pressure to obtain compound 1-3. Compound 1-3 was used directly in the synthesis of compound 1-4 without purification. Compound 1-3 was a yellow oil (1.35 g, yield 99%). The mass spectrometric data of compound 1-3 were: ESI-MS (m / z): 383.11 [M+H] + .

[0138] Step 4: Compound 1-3 (1.30 g, 1.00 eq) was dissolved in N, N-dimethylformamide (10 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (1.00 g, 1.05 eq, CAS No.: 81836-43-3) and N, N-diisopropylethylamine (1.32 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.55 g, 1.2 eq, CA The reaction mixture was stirred at 25°C for 16 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was diluted with saturated aqueous sodium carbonate (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 1-4. Compound 1-4 was obtained as a yellow oil (1.73 g, yield 79%). The mass spectrometric data of compound 1-4 were: ESI-MS (m / z): 645.25 [M+H] + .

[0139] Step 5: Compound 1-4 (1.73 g, 1.00 eq) was dissolved in a mixture of methanol / dichloromethane / tetrahydrofuran (50 mL / 40 mL / 30 mL) to obtain a solution. 5% palladium on carbon (0.30 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 24 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 1-5. Compound 1-5 was obtained as a yellow oil (0.92 g, yield 65%). The mass spectrum data of compound 1-5 is: ESI-MS (m / z): 527.25 [M+H] + .

[0140] Step 6: Compound 1-5 (0.92 g, 1.0 eq) was dissolved in dichloromethane (20 mL) to obtain a solution. Triethylamine (0.35 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.32 g, 1.2 eq, CAS No. 55750-48-6) were added to the solution, and the mixture was heated to 45°C and reacted at 45°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 1-6. Compound 1-6 was obtained as a yellow oil (0.79 g, 74% yield). The mass spectrum data of compound 1-6 is: ESI-MS (m / z): 607.26 [M+H] + .

[0141] Step 7: Compound 1-6 (30 mg, 1.00 eq) was dissolved in N,N-dimethylformamide (1.5 mL) to obtain a solution; Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS No.: 1599440-12-6) (41 mg, 1.00 eq) and N,N-diisopropylethylamine (32 mL) were added to the solution. After the reaction mixture was cooled to 0°C, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (37.6 mg, 2.00 eq) was added, and the mixture was reacted at 25°C for 2 hours to obtain a reaction solution. After the reaction was completed under HPLC monitoring, the reaction solution was purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-1. Compound LP-1 is a yellow powdery solid (22 mg, yield 31%). The synthetic route of compound LP-1 is shown in the following table. Figure 4 The mass spectrometry results of compound LP-1 are shown in Figure 5 The mass spectrum data of compound LP-1 are: LCMS (ESI) [M+H] + : 1429.55[M+H] + .

[0142] Example 2-2: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-2, wherein the linker-cytotoxin LP-2 has the following structure: .

[0143] The preparation method of the linker-cytotoxin LP-2 comprises the following steps: Step 1: Compound 4-bromo-2-fluorobenzaldehyde (2.00 g, 1.00 eq) was dissolved in N,N-dimethylformamide (20 mL) to obtain a solution; N-Boc-piperazine (1.6 g, 1.2 eq) and potassium carbonate (1.2 g, 1.2 eq) were added to the solution in sequence, stirred to mix, and then stirred at 100°C for 24 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature (25°C), then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL×3), and the organic phase was collected; the organic phase was first washed with saturated brine (40 mL×1), then concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 2-1. Compound 2-1 was a yellow oil (2.14 g, yield 78%). The mass spectrum data of compound 2-1 was: ESI-MS (m / z): 369.02 [M+H] + .

[0144] Step 2: Compound 2-1 (2.14 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (3.42 g, 1.50 eq, CAS No. 15097-38-8) was added to the solution, stirred evenly, and reacted at 20°C for 18 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 2-2. Compound 2-2 was obtained as a yellow oil (2.78 g, yield 96%). The mass spectrometric data of compound 2-2 were: ESI-MS (m / z): 501.12 [M+H] + .

[0145] Step 3: Compound 2-2 (2.78 g, 1.00 eq) was dissolved in toluene (30 mL) to obtain a solution; benzyl carbamate (0.94 g, 1.20 eq), palladium acetate (23 mg, 0.025 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.12 g, 0.05 eq, CAS No.: 564483-18-7) and potassium carbonate (0.85 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and heated at 10 The reaction was continued at 0°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 2-3. Compound 2-3 was obtained as a yellow oil (3.1 g, yield 96%). The mass spectrometric data of compound 2-3 were: ESI-MS (m / z): 572.23 [M+H] + .

[0146] Step 4: Dissolve compound 2-3 (3.1 g, 1.00 eq) in ethyl acetate (10 mL) to obtain a solution. After cooling the solution to 0°C, add hydrogen chloride in ethyl acetate (4 mol / L, 20 mL, 15 eq) to the solution, stir evenly, and then stir at 0°C for 24 hours to obtain a reaction solution. After thin-layer chromatography (TLC) shows the reaction is complete, filter the reaction solution, dissolve the filter cake in 30 mL of a mixture of dichloromethane / methanol (dichloromethane:methanol = 10:1, v / v), and collect the organic phase. Wash the organic phase with saturated sodium carbonate aqueous solution (10 mL x 2) and evaporate to dryness under reduced pressure to obtain compound 2-4. Compound 2-4 was used directly in the synthesis of compound 2-5 without purification. Compound 2-4 is a yellow oil (1.6 g, 62% yield). The mass spectrometric data of compound 2-4 are: ESI-MS (m / z): 472.25 [M+H] + .

[0147] Step 5: Compound 2-4 (0.8 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (0.52 g, 1.1 eq) and N, N-diisopropylethylamine (0.66 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.77 g, 1.2 eq) was added, and then The reaction was stirred at 25°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was diluted with saturated aqueous sodium carbonate (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and finally purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 2-5. Compound 2-5 was obtained as a yellow oil (0.95 g, 76% yield). The mass spectrometric data of compound 2-5 were: ESI-MS (m / z): 734.33 [M+H] + .

[0148] Step 6: Compound 2-5 (0.95 g, 1.00 eq) was dissolved in a mixture of dichloromethane and methanol (2 mL / 10 mL) to obtain a solution. 5% palladium on carbon (0.09 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 12 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 2-6. Compound 2-6 was obtained as a yellow oil (0.43 g, yield 65%). The mass spectrometric data of compound 2-6 were: ESI-MS (m / z): 512.21 [M+H] + .

[0149] Step 7: Compound 2-6 (0.15 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.06 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.06 g, 1.2 eq) were added to the solution, and the temperature was raised to 45°C and allowed to react at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 2-7. Compound 2-7 was obtained as a yellow oil (0.12 g, yield 64%). The mass spectrometric data of compound 2-7 were: ESI-MS (m / z): 592.20 [M+H] + .

[0150] Step 8: Compound 2-7 (40 mg, 1.20 eq) was dissolved in N,N-dimethylformamide (0.5 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (50 mg, 1.00 eq) and N,N-diisopropylethylamine (22 mg, 3.00 eq) were added to the solution, and the mixture was cooled to 0°C. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (33 mg, 1.50 eq) was then added. The mixture was reacted at 25°C for 1 hour to obtain a reaction solution. After the reaction was completed by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-2. Compound LP-2 was obtained as a yellow powdery solid (20 mg, 20% yield). The synthetic route of compound LP-2 is shown in Figure 6 The mass spectrometry results of compound LP-2 are shown in Figure 7 The mass spectrum data of compound LP-2 are: LCMS (ESI) [M+H] + :1414.40[M+H] + .

[0151] Example 2-3: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-3, wherein the linker-cytotoxin LP-3 has the following structure: .

[0152] The preparation method of the linker-cytotoxin LP-3 comprises the following steps: Step 1: Dissolve the compound 4-bromo-2-fluorobenzaldehyde (1.5 g, 1.00 eq) in N,N-dimethylformamide (15 mL) to obtain a solution; add 4-(N-Boc-amino)piperidine (1.28 g, 1.2 eq, CAS No.: 73874-95-0) and potassium carbonate (0.88 g, 1.2 eq) to the solution in sequence, stir to mix, and then stir at 100 ° C for 24 hours to obtain a reaction solution; thin layer chromatography After HPLC indicated the reaction was complete, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated brine (30 mL x 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 3-1. Compound 3-1 was obtained as a yellow oil (2 g, yield 70%). The mass spectrometric data of compound 3-1 were: ESI-MS (m / z): 383.03 [M+H] + .

[0153] Step 2: Compound 3-1 (2 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (3.5 g, 1.60 eq) was added to the solution, and the mixture was stirred at 20°C for 18 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 3-2. Compound 3-2 was obtained as a yellow oil (2.57 g, yield 95%). The mass spectrometric data of compound 3-2 were: ESI-MS (m / z): 515.18 [M+H] + .

[0154] Step 3: Compound 3-2 (2.5 g, 1.00 eq) was dissolved in toluene (30 mL) to obtain a solution; benzyl carbamate (0.88 g, 1.20 eq), palladium acetate (27 mg, 0.025 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.11 g, 0.05 eq) and potassium carbonate (0.8 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and the reaction was continued at 100 ° C for 4 hours to obtain a reaction solution After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C) and concentrated under reduced pressure to obtain a residue. The residue was diluted with water (40 mL) and extracted with dichloromethane (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and finally purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 3-3. Compound 3-3 was obtained as a yellow oil (2.27 g, yield 80%). The mass spectrometric data of compound 3-3 were: ESI-MS (m / z): 586.31 [M+H] + .

[0155] Step 4: Compound 3-3 (2.2 g, 1.00 eq) was dissolved in ethyl acetate (8 mL) to obtain a solution; after adding hydrogen chloride ethyl acetate solution (4 mol / L, 14 mL, 15 eq) to the solution, the temperature was lowered to 0 ° C and the reaction was continued at 0 ° C for 24 hours to obtain a reaction solution; after the reaction was completed by thin layer chromatography, the reaction solution was concentrated under reduced pressure to obtain a residue; the reaction solution was first diluted with saturated sodium carbonate aqueous solution (30 mL), then extracted with ethyl acetate (30 mL × 3), and the organic phase was taken; the organic phase was first washed with saturated brine (30 mL × 3), and then evaporated to dryness under reduced pressure to obtain compound 3-4. Compound 3-4 was not purified and was directly used in the synthesis of compound 3-5. Compound 3-4 is a yellow oil (1.72 g, yield 94%). The mass spectrometry data of compound 3-4 is: ESI-MS (m / z): 486.24 [M+H] + .

[0156] Step 5: Compound 3-4 (1.7 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (1.1 g, 1.1 eq) and N, N-diisopropylethylamine (1.35 g, 3.00 eq) were added to the solution, and the mixture was cooled to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.6 g, 1.2 eq) was added. The mixture was stirred and reacted at 25°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 3), concentrated under reduced pressure, and finally purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 3-5. Compound 3-5 was obtained as a yellow oil (1.2 g, yield 46%). The mass spectrometric data of compound 3-5 were: ESI-MS (m / z): 748.24 [M+H] + .

[0157] Step 6: Dissolve compound 3-5 (1.2 g, 1.00 eq) in methanol (10 mL) to obtain a solution; add 5% palladium carbon (0.1 g) to the solution, and react at 25 ° C for 12 hours under a hydrogen atmosphere to obtain a reaction solution; thin layer chromatography shows that the reaction is complete, filter the reaction solution, and take the filter cake; rinse the filter cake with methanol (5 mL) and take the filtrate; evaporate the filtrate to dryness under reduced pressure to obtain compound 3-6. Compound 3-6 is not purified and is directly used in the synthesis of compound 3-7. Compound 3-6 is a yellow oil (0.80 g, yield 95%). The mass spectrometry data of compound 3-6 are: ESI-MS (m / z): 526.30 [M+H] + .

[0158] Step 7: Compound 3-6 (0.2 g, 1.00 eq) was dissolved in dichloromethane (10 mL) to obtain a solution. Triethylamine (0.077 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.071 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 3-7. Compound 3-7 was obtained as a yellow oil (0.16 g, yield 69%). The mass spectrometric data of compound 3-7 were: ESI-MS (m / z): 606.20 [M+H] + .

[0159] Step 8: Compound 3-7 (38 mg, 1.10 eq) was dissolved in N,N-dimethylformamide (1 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (50 mg, 1.00 eq), N,N-diisopropylethylamine (22 mg, 3.00 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (33 mg, 1.10 eq), and 1-hydroxybenzotriazole (8.5 mg, 1.10 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-3. Compound LP-3 was obtained as a yellow powdery solid (48 mg, 59% yield). The synthetic route of compound LP-3 is shown in Figure 8 The mass spectrometry results of compound LP-3 are shown in Figure 9 The mass spectrum data of compound LP-2 are: LCMS (ESI) [M+H] + :1428.45[M+H] + .

[0160] Example 2-4: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-4, wherein the linker-cytotoxin LP-4 has the following structure: .

[0161] The preparation method of the linker-cytotoxin LP-4 comprises the following steps: Step 1: Dissolve 4-bromo-2-fluorobenzaldehyde (1.5 g, 1.00 eq) in N,N-dimethylformamide (10 mL) to obtain a solution. Add diethylamine (0.66 g, 1.2 eq) and potassium carbonate (0.9 g, 1.2 eq) to the solution in sequence, stir to mix, and then react at 100°C for 24 hours to obtain a reaction solution. After thin-layer chromatography (TLC) shows completion of the reaction, cool the reaction solution to room temperature (25°C), dilute with water (40 mL), and extract with ethyl acetate (30 mL x 3). The organic phase is washed with saturated brine (30 mL x 1), concentrated under reduced pressure, and purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 4-1. Compound 4-1 is a yellow oil (1.5 g, yield 79%). The mass spectrum data of compound 4-1 is: ESI-MS (m / z): 256.01 [M+H] + . 1 H NMR (400 MHz, Chloroform- d ) δ 10.25 (s, 1H), 7.67(d, J = 8.3 Hz, 1H), 7.29 (d, J = 1.7 Hz, 1H), 7.22 (dd, J = 8.3, 1.7 Hz, 1H), 3.22(q, J = 7.1 Hz, 4H), 1.11 (t, J = 7.1 Hz, 6H). Step 2: Compound 4-1 (1.46 g, 1.00 eq) was dissolved in dichloromethane (20 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (3.5 g, 1.50 eq) was added to the solution, stirred evenly, and reacted at 20°C for 18 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 4-2. Compound 4-2 was obtained as a yellow oil (2.12 g, yield 95%). The mass spectrometric data of compound 4-2 were: ESI-MS (m / z): 388.11 [M+H] + . 1 H NMR (400 MHz, Chloroform- d ) δ 8.06(d, J= 16.2 Hz, 1H), 7.48 – 7.30 (m, 7H), 7.23 – 7.02 (m, 2H), 6.43 (d, J = 16.2Hz, 1H), 5.28 (s, 2H), 3.07 (q, J = 7.1 Hz, 4H), 1.03 (t, J = 7.1 Hz, 6H). Step 3: Compound 4-2 (2.12 g, 1.00 eq) was dissolved in toluene (20 mL) to obtain a solution; benzyl carbamate (1 g, 1.20 eq), palladium acetate (31 mg, 0.025 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.13 g, 0.05 eq) and potassium carbonate (0.91 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and the reaction was continued at 100 ° C for 4 hours to obtain The reaction mixture was cooled to room temperature (25°C) and concentrated under reduced pressure after thin-layer chromatography (TLC) showed the reaction was complete. The mixture was then diluted with water (40 mL) and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated brine (30 mL x 1), concentrated under reduced pressure, and purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 4-3. Compound 4-3 was obtained as a yellow oil (2.03 g, yield 80%). The mass spectrometric data of compound 4-3 were: ESI-MS (m / z): 459.18 [M+H] + .

[0162] Step 4: Dissolve compound 4-3 (2.35 g, 1.00 eq) in methanol (20 mL) to obtain a solution; add 5% palladium carbon (0.2 g) to the solution, and react at 25 ° C for 12 hours under a hydrogen atmosphere to obtain a reaction solution; after thin layer chromatography shows that the reaction is complete, filter the reaction solution and take the filter cake; rinse the filter cake with methanol (5 mL) and take the filtrate; evaporate the filtrate to dryness under reduced pressure to obtain compound 4-4. Compound 4-4 is not purified and is directly used in the synthesis of compound 4-5. Compound 4-4 is a yellow oil (1.15 g, yield 95%). The mass spectrometry data of compound 4-4 are: ESI-MS (m / z): 237.11 [M+H] + .

[0163] Step 5: Compound 4-4 (0.1 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.084 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.078 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), then adjusted to pH 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 4-5. Compound 4-5 was obtained as a yellow oil (0.09 g, yield 69%). The mass spectrum data of compound 4-5 is: ESI-MS (m / z): 317.09 [M+H] + .

[0164] Step 6: Compound 4-5 (56 mg, 2.60 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (60 mg, 1.00 eq), N,N-diisopropylethylamine (22 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (46.2 mg, 1.30 eq), and 1-hydroxybenzotriazole (4.6 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-4. Compound LP-4 was obtained as a yellow powdery solid (20 mg, 25% yield). The synthetic route of compound LP-4 is shown in Figure 10 The mass spectrometry results of compound LP-4 are shown in Figure 11 The mass spectrum data of compound LP-4 are: LCMS (ESI) [M+H] + : 1139.46[M+H] + .

[0165] Example 2-5: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-5, wherein the linker-cytotoxin LP-5 has the following structure: .

[0166] The preparation method of the linker-cytotoxin LP-5 comprises the following steps: Step 1: Compound 4-bromo-2-fluorobenzaldehyde (2 g, 1.00 eq) was dissolved in N,N-dimethylformamide (20 mL) to obtain a solution; N-methylpiperazine (1.18 g, 1.2 eq) and potassium carbonate (1.63 g, 1.2 eq) were added to the solution in sequence, stirred to mix, and then stirred at 100°C for 24 hours to obtain a reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature (25°C), then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL×3), and the organic phase was collected; the organic phase was first washed with saturated brine (30 mL×1), then concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-1. Compound 5-1 was a yellow oil (2.17 g, yield 78%). The mass spectrum data of compound 5-1 was: ESI-MS (m / z): 283.01 [M+H] + .

[0167] Step 2: Compound 5-1 (2.14 g, 1.00 eq) was dissolved in dichloromethane (25 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (4.7 g, 1.50 eq) was added to the solution and stirred until uniform. Finally, the mixture was reacted at 20°C for 18 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether to ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-2. Compound 5-2 was obtained as a yellow oil (2.12 g, yield 67%). The mass spectrometric data of compound 5-2 were: ESI-MS (m / z): 415.11 [M+H] + .

[0168] Step 3: Compound 5-2 (2.1 g, 1.00 eq) was dissolved in toluene (20 mL) to obtain a solution; benzyl carbamate (0.78 g, 1.20 eq), palladium acetate (48 mg, 0.05 eq), 2-dicyclohexylphosphine-2', 4', 6'-triisopropylbiphenyl (0.2 g, 0.1 eq) and potassium carbonate (0.72 g, 1.2 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C under a nitrogen atmosphere and the reaction was continued at 100 ° C for 4 hours. A reaction solution was obtained. After the reaction was completed by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), diluted with water (40 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with saturated brine (30 mL × 1), concentrated under reduced pressure, and finally purified by silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 5-3. Compound 5-3 was obtained as a yellow oil (1.08 g, yield 44%). The mass spectrometric data of compound 5-3 were: ESI-MS (m / z): 486.25 [M+H] + .

[0169] Step 4: Dissolve compound 5-3 (1.08 g, 1.00 eq) in methanol (20 mL) to obtain a solution; add 5% palladium carbon (0.2 g) to the solution, and react at 25 ° C for 12 hours under a hydrogen atmosphere to obtain a reaction solution; after thin layer chromatography shows that the reaction is complete, filter the reaction solution and take the filter cake; rinse the filter cake with methanol (5 mL) and take the filtrate; evaporate the filtrate to dryness under reduced pressure to obtain compound 5-4. Compound 5-4 is not purified and is directly used in the synthesis of compound 5-5. Compound 5-4 is a yellow oil (0.59 g, yield 100%). The mass spectrometry data of compound 5-4 is: ESI-MS (m / z): 264.15 [M+H] + .

[0170] Step 5: Compound 5-4 (0.05 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution; triethylamine (0.038 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.035 g, 1.2 eq) were added to the solution, stirred and mixed, and then reacted at 45°C for 4 hours to obtain a reaction solution; after the reaction was completed by thin layer chromatography, the reaction solution was cooled to room temperature (25°C) and then evaporated to dryness under reduced pressure to obtain compound 5-5. Compound 5-5 was not purified and was directly used in the synthesis of compound 5-6. Compound 5-6 was a yellow oil (0.06 g, yield 92%). The mass spectrometry data was: ESI-MS (m / z): 344.09 [M+H] + .

[0171] Step 6: Compound 5-5 (65 mg, 2.70 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (60 mg, 1.00 eq), N,N-diisopropylethylamine (22 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (46.2 mg, 1.30 eq), and 1-hydroxybenzotriazole (4.6 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-5. Compound LP-5 was obtained as a yellow powdery solid (17 mg, 21% yield). The synthetic route of compound LP-5 is shown in Figure 12 The mass spectrometry results of compound LP-5 are shown in Figure 13 The mass spectrum data of compound LP-5 are: LCMS (ESI) [M+H] + : 1166.47[M+H] + .

[0172] Example 2-6: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-6, wherein the linker-cytotoxin LP-6 has the following structure: .

[0173] The preparation method of the linker-cytotoxin LP-6 comprises the following steps: Step 1: Compound 2-4 (0.2 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; N-Fmoc-glycyl-glycine (0.18 g, 1.2 eq) and N, N-diisopropylethylamine (0.14 g, 2.5 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.19 g, 1 .2eq), and then stirred at 25 ° C for 4 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30 mL), then extracted with ethyl acetate (30 mL × 3), and the organic phase was taken; the organic phase was first washed and diluted with saturated sodium bicarbonate solution (30 mL), then washed with saturated brine (30 mL × 3), and then evaporated to dryness under reduced pressure to obtain compound 6-1. Compound 6-1 was not purified and was directly used in the synthesis of compound 6-2. Compound 6-1 is a yellow oil (0.36 g, 100%). The mass spectrometry data of compound 6-1 is: ESI-MS (m / z): 808.26 [M+H] + .

[0174] Step 2: Compound 6-1 (0.36 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. The solution was cooled to 0°C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.068 g, 1.00 eq) was added to the solution. The mixture was then reacted at 0°C for 1.5 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 6-2. Compound 6-2 was obtained as a yellow oil (0.21 g, 80% yield). The mass spectrometric data of compound 6-2 were: ESI-MS (m / z): 586.21 [M+H] + .

[0175] Step 3: Compound 6-2 (0.21 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 4,7,10,13,16-pentaoxaheptadecanoic acid (0.12 g, 1.2 eq) and N, N-diisopropylethylamine (0.12 g, 2.50 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.16 g, 1.2 eq) was added, and then the mixture was stirred at 2 ° C for 2 hours. The reaction was stirred at 5°C for 2 hours to obtain a reaction solution. After thin-layer chromatography indicated the reaction was complete, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 5). The organic phase was washed with water (30 mL) and then with saturated brine (30 mL), then concentrated under reduced pressure and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 6-3. Compound 6-3 was obtained as a yellow oil (0.19 g, yield 62.5%). The mass spectrometric data of compound 6-3 were: ESI-MS (m / z): 848.37 [M+H] + .

[0176] Step 4: Compound 6-3 (0.19 g, 1.00 eq) was dissolved in methanol (5 mL) to obtain a solution. 5% palladium on carbon (0.02 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 6-4. Compound 6-4 was obtained as a yellow oil (0.06 g, yield 43%). The mass spectrometric data of compound 6-4 were: ESI-MS (m / z): 626.39 [M+H] + .

[0177] Step 5: Compound 6-4 (0.06 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.019 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.018 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 6-5. Compound 6-5 was obtained as a yellow oil (0.02 g, 30% yield). The mass spectrometric data of compound 6-5 were: ESI-MS (m / z): 706.29 [M+H] + .

[0178] Step 6: Compound 6-5 (120 mg, 2.20 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (75 mg, 1.00 eq), N,N-diisopropylethylamine (27.6 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (48.9 mg, 1.10 eq), and 1-hydroxybenzotriazole (5.8 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-6. Compound LP-6 was obtained as a yellow powdery solid (55 mg, 39% yield). The synthetic route of compound LP-6 is shown in Figure 14 The mass spectrometry results of compound LP-6 are shown in Figure 15 The mass spectrum data of compound LP-6 are: LCMS (ESI) [M+H] + : 1528.64[M+H] + .

[0179] Example 2-7: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-7, wherein the linker-cytotoxin LP-7 has the following structure: .

[0180] The preparation method of the linker-cytotoxin LP-7 comprises the following steps: Step 1: Compound 2-4 (1.3 g, 1.00 eq) was dissolved in N, N-dimethylformamide (10 mL) to obtain a solution; 4,7,10,13,16,19,22,25,28-nonaoxaconicoic acid (1.39 g, 1.1 eq) and N, N-diisopropylethylamine (1.07 g, 3.00 eq) were added to the solution, and the mixture was cooled to 0 ° C, and then 2-(7-azabenzotriazole)-N , N,N',N'-tetramethyluronium hexafluorophosphate (1.26g, 1.2eq), and then reacted at 25°C for 8 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30mL), then extracted with ethyl acetate (30mL×3), and the organic phase was taken; the organic phase was first washed with sodium bicarbonate solution (30mL), then washed with saturated brine (30mL), and then evaporated to dryness under reduced pressure to obtain compound 7-1. Compound 7-1 was not purified and was directly used in the synthesis of compound 7-2. Compound 7-1 is a yellow oil (2.51g, yield 100%). The mass spectrometry data of compound 7-1 are: ESI-MS (m / z): 910.51[M+H] + .

[0181] Step 2: Compound 7-1 (2.51 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.25 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 7-2. Compound 7-2 was obtained as a yellow oil (1.6 g, yield 83%). The mass spectrometric data of compound 7-2 were: ESI-MS (m / z): 688.31 [M+H] + .

[0182] Step 3: Compound 7-2 (0.35 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.103 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.095 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 7-3. Compound 7-3 was obtained as a yellow oil (0.35 g, yield 89%). The mass spectrometric data of compound 7-3 were: ESI-MS (m / z): 768.31 [M+H] + .

[0183] Step 4: Compound 7-3 (130 mg, 2.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (75 mg, 1.00 eq), N,N-diisopropylethylamine (27.6 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (48.9 mg, 1.10 eq), and 1-hydroxybenzotriazole (5.8 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-7. Compound LP-7 was obtained as a yellow powdery solid (70 mg, 48% yield). The synthetic route of compound LP-7 is shown in Figure 16 The mass spectrometry results of compound LP-7 are shown in Figure 17 The mass spectrum data of compound LP-7 are: LCMS (ESI) [M+H] + : 1590.70[M+H] + .

[0184] Example 2-8: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-8, wherein the linker-cytotoxin LP-8 has the following structure: .

[0185] The preparation method of the linker-cytotoxin LP-8 comprises the following steps: Step 1: Compound 3-4 (1.97 g, 1.00 eq) was dissolved in N, N-dimethylformamide (25 mL) to obtain a solution; 4,7,10,13,16,19,22,25,28-nonaoxaconicoic acid (2.04 g, 1.1 eq) and N, N-diisopropylethylamine (1.73 g, 3.00 eq) were added to the solution, and the temperature was first lowered to 0 ° C, and then 2-(7-azabenzotriazole)-N , N,N',N'-tetramethyluronium hexafluorophosphate (1.85g, 1.2eq), and then reacted at 25°C for 8 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first diluted with water (30mL), then extracted with ethyl acetate (30mL×3), and the organic phase was taken; the organic phase was first washed with saturated sodium bicarbonate solution (30mL), then washed with saturated brine (30mL), and then evaporated to dryness under reduced pressure to obtain compound 8-1. Compound 8-1 is a yellow oil (3g, yield 80%). Compound 8-1 was not purified and was directly used in the synthesis of compound 8-2. The mass spectrometry data of compound 8-1 is: ESI-MS (m / z): 924.42[M+H] + .

[0186] Step 2: Compound 8-1 (3 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.3 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 8-2. Compound 8-2 was obtained as a yellow oil (1.9 g, yield 83%). The mass spectrometric data of compound 8-2 were: ESI-MS (m / z): 702.39 [M+H] + .

[0187] Step 3: Compound 8-2 (0.3 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.08 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.086 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 8-3. Compound 8-3 was obtained as a yellow oil (0.24 g, 72% yield). The mass spectrometric data of compound 8-3 were: ESI-MS (m / z): 782.46 [M+H] + .

[0188] Step 4: Compound 8-3 (185 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (70 mg, 1.00 eq), N,N-diisopropylethylamine (25.5 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (45 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-8. Compound LP-8 was obtained as a yellow powdery solid (75 mg, 58% yield). The synthetic route of compound LP-8 is shown in Figure 18 The mass spectrometry results of compound LP-8 are shown in Figure 19 The mass spectrum data of compound LP-8 are: LCMS (ESI) [M+H] + : 1604.72[M+H] + .

[0189] Example 2-9: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-9, wherein the linker-cytotoxin LP-9 has the following structure: .

[0190] The preparation method of the linker-cytotoxin LP-9 comprises the following steps: Step 1: Compound 2-4 (1 g, 1.00 eq) was dissolved in N,N-dimethylformamide (10 mL) to obtain a solution. Methyl-pentaethylene glycol monobromide (0.75 g, 1.1 eq) and potassium carbonate (0.44 g, 1.5 eq) were added to the solution, stirred uniformly, then heated to 70°C and reacted at 70°C for 8 hours to obtain a reaction solution. After thin-layer chromatography (TLC) indicated completion of the reaction, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL x 3). The organic phase was collected and washed with saturated brine (30 mL) and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 9-1. Compound 9-1 was obtained as a yellow oil (1.1 g, yield 73%). The mass spectrum data of compound 9-1 is: ESI-MS (m / z): 706.29 [M+H] + .

[0191] Step 2: Compound 9-1 (1.1 g, 1.00 eq) was dissolved in methanol (10 mL) to obtain a solution. 5% palladium on carbon (0.1 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 22 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 9-2. Compound 9-2 was obtained as a yellow oil (0.35 g, 46% yield). The mass spectrometric data of compound 9-2 were: ESI-MS (m / z): 484.31 [M+H] + .

[0192] Step 3: Compound 9-2 (0.2 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.08 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.08 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 9-3. Compound 9-3 was obtained as a yellow oil (0.23 g, yield 98%). The mass spectrometric data of compound 9-3 were: ESI-MS (m / z): 564.23 [M+H] + .

[0193] Step 4: Compound 9-3 (100 mg, 2.40 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (65 mg, 1.00 eq), N,N-diisopropylethylamine (24 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-9. Compound LP-9 was obtained as a yellow powdery solid (36 mg, 32% yield). The synthetic route of compound LP-9 is shown in Figure 20 The mass spectrometry results of compound LP-9 are shown in Figure 21 The mass spectrum data of compound LP-9 are: LCMS (ESI) [M+H] + :1386.60[M+H] + .

[0194] Example 2-10: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-10, wherein the linker-cytotoxin LP-10 has the following structure: .

[0195] The preparation method of the linker-cytotoxin LP-10 comprises the following steps: Step 1: Compound 6-2 (1.6 g, 1.00 eq) was dissolved in N, N-dimethylformamide (10 mL) to obtain a solution; 4,7,10,13,16,19,22,25,28-nonaoxaconicoic acid (1.49 g, 1.2 eq) and N, N-diisopropylethylamine (0.88 g, 2.5 eq) were added to the solution, and the mixture was cooled to 0 ° C, and then 2-(7-azabenzotriazole) was added. -N,N,N',N'-tetramethyluronium hexafluorophosphate (1.28 g, 1.2 eq) was added and reacted at 25°C for 2 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 5). The organic phase was collected and washed with water (30 mL) and then with saturated brine (30 mL), and then evaporated to dryness under reduced pressure to obtain compound 10-1. Compound 10-1 was used directly in the synthesis of compound 10-2 without purification. Compound 10-1 was a yellow oil (2.7 g, yield 96%). The mass spectrometric data of compound 10-1 were: ESI-MS (m / z): 1024.46 [M+H] + .

[0196] Step 2: Compound 10-1 (2.8 g, 1.00 eq) was dissolved in methanol (15 mL) to obtain a solution. 5% palladium on carbon (0.3 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with a mixture of dichloromethane and methanol (5 mL / 5 mL), and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 10-2. Compound 10-2 was obtained as a yellow oil (1.66 g, yield 75%). The mass spectrometric data of compound 10-2 were: ESI-MS (m / z): 802.36 [M+H] + .

[0197] Step 3: Compound 10-2 (0.05 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.013 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.012 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After thin-layer chromatography indicated completion of the reaction, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 10-3. Compound 10-3 was obtained as a yellow oil (0.02 g, 36% yield). The mass spectrum data of compound 10-3 is: ESI-MS (m / z): 882.35 [M+H] + .

[0198] Step 4: Compound 10-3 (150 mg, 2.30 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (65 mg, 1.00 eq), N,N-diisopropylethylamine (24 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The temperature was then cooled to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-10. Compound LP-10 was obtained as a yellow powdery solid (84 mg, 62% yield). The synthetic route of compound LP-10 is shown in Figure 22 The mass spectrometry results of compound LP-10 are shown in Figure 23 The mass spectrum data of compound LP-10 are: LCMS (ESI) [M+H] + : 1705.74[M+H] + .

[0199] Example 2-11: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-11, wherein the linker-cytotoxin LP-11 has the following structure: .

[0200] The preparation method of the linker-cytotoxin LP-11 comprises the following steps: Step 1: Compound 7-3 (92 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS No.: CAS2845164-91-0) (30 mg, 1.00 eq), N,N-diisopropylethylamine (12.6 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (22.8 mg, 1.10 eq) and 1-hydroxybenzotriazole (2.4 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-11. Compound LP-11 is a yellow powdery solid (39 mg, yield 65%). The synthetic route of compound LP-11 is shown in Figure 24 The mass spectrometry results of compound LP-11 are shown in Figure 25 The mass spectrum data of compound LP-11 are: LCMS (ESI) [M+H] + : 1504.69[M+H] + .

[0201] Example 2-12: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-12, wherein the linker-cytotoxin LP-12 has the following structure: .

[0202] The preparation method of the linker-cytotoxin LP-12 comprises the following steps: Step 1: Compound 8-3 (94 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) (30 mg, 1.00 eq), N,N-diisopropylethylamine (12.8 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (22.8 mg, 1.10 eq), and 1-hydroxybenzotriazole (2.4 mg, 0.50 eq) were added to the solution. The mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-12. Compound LP-12 was obtained as a yellow powdery solid (48 mg, 79% yield). The synthetic route of compound LP-12 is shown in Figure 26 The mass spectrometry results of compound LP-12 are shown in Figure 27 The mass spectrum data of compound LP-12 are: LCMS (ESI) [M+H] + : 1518.55[M+H] + .

[0203] Example 2-13: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-13, wherein the linker-cytotoxin LP-13 has the following structure: .

[0204] The preparation method of the linker-cytotoxin LP-13 comprises the following steps: Step 1: Dissolve the compound N-[(1,1-dimethylethoxy)carbonyl]glycylglycyl-L-phenylalanyl-glycine (purchased from MedChemExpress, CAS No.: 187794-49-6) (0.25 g, 1.20 eq) in N,N-dimethylformamide (5 mL) to obtain a solution; add isotecan mesylate (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS: 169869-90-3) (0.25 g, 1.00 eq), N,N-diisopropylamine, 1,2-dimethylthiazolinone ... Propylethylamine (0.216 g, 3.50 eq) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (0.30 g, 1.20 eq) were added, cooled to 0°C, and reacted at 0°C for 1 hour to obtain a reaction solution. After HPLC monitoring of the reaction completion, the reaction solution was evaporated to dryness under reduced pressure, then redissolved in dichloromethane to the original volume, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 11-1. Compound 11-1 was obtained as a yellow solid (0.4 g, 98% yield). The mass spectrometric data of compound 11-1 were: ESI-MS (m / z): 854.32 [M+H] + .

[0205] Step 2: Dissolve compound 11-1 (0.4 g, 1.00 eq) in dioxane (2 mL) to obtain a solution. Add hydrogen chloride in ethyl acetate (4 mol / L, 3 mL, 25.6 eq) to the solution, stir evenly, and then react at room temperature (25°C) for 14 hours to obtain a reaction solution. After LC-MS monitoring shows the reaction is complete, filter the reaction solution to obtain a filter cake. Rinse the filter cake with ethyl acetate (5 mL) and obtain a filtrate. The filtrate is dried to obtain compound 11-2. Compound 11-2 is a yellow solid (0.34 g, yield 92%). The mass spectrometric data of compound 11-2 are: ESI-MS (m / z): 754.30 [M+H] + .

[0206] Step 3: Compound 8-3 (156 mg, 3.00 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; compound 11-2 (50 mg, 1.00 eq), N,N-diisopropylethylamine (30 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.20 eq) and 1-hydroxybenzotriazole (4.5 mg, 0.50 eq) were added to the solution, and the temperature was lowered to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-13. Compound LP-13 was obtained as a white powdery solid (27 mg, yield 28%). The synthetic route of compound LP-13 is shown in Figure 28 The mass spectrometry results of compound LP-13 are shown in Figure 29 The mass spectrum data of compound LP-13 are: LCMS (ESI) [M+H] + : 1517.69[M+H] + .

[0207] Example 2-14: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-14, wherein the linker-cytotoxin LP-14 has the following structure: .

[0208] The preparation method of the linker-cytotoxin LP-14 comprises the following steps: Step 1: Compound 10-3 (100 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (1 mL) to obtain a solution. Compound 11-2 (36 mg, 1.00 eq), N,N-diisopropylethylamine (18 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (26 mg, 1.10 eq), and 1-hydroxybenzotriazole (3 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed by HPLC monitoring, the reaction solution was purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-14. Compound LP-14 was obtained as a white powdery solid (18 mg, yield 24%). The synthetic route of compound LP-14 is shown in Figure 30 The mass spectrometry results of compound LP-14 are shown in Figure 31 The mass spectrum data of compound LP-14 are: LCMS (ESI) [M / 2+H]+ :809.36 [M / 2+H] + .

[0209] Example 2-15: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-15, wherein the linker-cytotoxin LP-15 has the following structure: .

[0210] The preparation method of the linker-cytotoxin LP-15 comprises the following steps: Step 1: Compound 3-4 (0.71 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioctadecane-38-oic acid (0.8 g, 1.00 eq, CAS No.: 2135793-73-4) and N, N-diisopropylethylamine (0.79 g, 4.50 eq) were added to the solution, and the temperature was first lowered to 0°C, then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.62 g, 1.2 eq) was added, and the mixture was reacted at 25°C for 8 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was collected and washed with saturated brine (30 mL × 3) and then evaporated to dryness under reduced pressure to obtain compound 12-1. Compound 12-1 was not purified and was directly used in the synthesis of compound 12-2. Compound 12-1 was a yellow oil (1.4 g, yield 100%, theoretical value). The mass spectrometric data of compound 12-1 were: ESI-MS (m / z): 1056.58 [M+H] + .

[0211] Step 2: Compound 12-1 (1.4 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution; 5% palladium on carbon (0.3 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 16 hours to obtain a reaction solution; after thin-layer chromatography showed that the reaction was complete, the reaction solution was filtered to obtain a filter cake; the filter cake was rinsed with a mixture of dichloromethane / methanol (dichloromethane:methanol = 1:1, v / v, 5 mL) and the filtrate was obtained; the filtrate was evaporated to dryness under reduced pressure to obtain compound 12-2. Compound 12-2 was a yellow oil (1.1 g, yield 100%, theoretical value). The mass spectrometry data of compound 12-2 were: ESI-MS (m / z): 834.50 [M+H] + .

[0212] Step 3: Compound 12-2 (0.6 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.15 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.13 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 12-3. Compound 12-3 was obtained as a yellow oil (0.6 g, 90% yield). The mass spectrometric data of compound 12-3 were: ESI-MS (m / z): 914.44 [M+H] + .

[0213] Step 4: Compound 12-3 (105 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; VA-PAB-Exatecan (35 mg, 1.00 eq), N,N-diisopropylethylamine (15 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (27 mg, 1.10 eq), and 1-hydroxybenzotriazole (3 mg, 0.50 eq) were added to the solution, and the temperature was cooled to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-15. Compound LP-15 was obtained as a yellow powdery solid (45 mg, yield 58%). The synthetic route of compound LP-15 is shown in Figure 32 The mass spectrometry results of compound LP-15 are shown in Figure 33 The mass spectrum data of compound LP-15 are: LCMS (ESI) [M / 2+H] + : 825.89[M / 2+H] + .

[0214] Example 2-16: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-16, wherein the linker-cytotoxin LP-16 has the following structure: .

[0215] The preparation method of the linker-cytotoxin LP-16 comprises the following steps: Step 1: Compound 12-3 (160 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; 11-2 (55 mg, 1.00 eq), N,N-diisopropylethylamine (31 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (73 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution; after HPLC monitoring of the reaction completion, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-16. Compound LP-16 was obtained as a white powdery solid (29 mg, yield 25%). The synthetic route of compound LP-16 is shown in Figure 34 The mass spectrometry results of compound LP-16 are shown in Figure 35 The mass spectrum data of compound LP-16 are: LCMS (ESI) [M+H] + :1649.47 [M+H] + .

[0216] Example 2-17: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-17, wherein the linker-cytotoxin LP-17 has the following structure: .

[0217] The preparation method of the linker-cytotoxin LP-17 comprises the following steps: Step 1: Compound 12-3 (63 mg, 2.50 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; GGFG-PAB-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS: 251459-32-2) (25 mg, 1.00 eq), N,N-diisopropylethylamine (9 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (18 mg, 1.25 eq) and 1-hydroxybenzotriazole (2 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution; after the reaction was completed by HPLC monitoring, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-17. Compound LP-17 is a white powdery solid (24 mg, yield 48%). The synthetic route of compound LP-17 is shown in Figure 36 The mass spectrometry results of compound LP-17 are shown in Figure 37The mass spectrum data of compound LP-17 are: LCMS (ESI) [M+H] + :1798.81 [M+H] + .

[0218] Example 2-18: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-18, wherein the linker-cytotoxin LP-18 has the following structure: .

[0219] The preparation method of the linker-cytotoxin LP-18 comprises the following steps: Step 1: Compound 2-4 (0.86 g, 1.00 eq) was dissolved in N, N-dimethylformamide (5 mL) to obtain a solution; 2,5,8,11,14,17,20,23,26,29,32,35-dodecatrioxane-38-oic acid (1.0 g, 1.00 eq) and N, N-diisopropylethylamine (0.54 g, 2.50 eq) were added to the solution, and the mixture was cooled to 0 ° C, and then 2 -(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.76 g, 1.2 eq) was added and reacted at 25°C for 8 hours to obtain a reaction solution. After thin-layer chromatography showed that the reaction was complete, the reaction solution was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The organic phase was collected and washed with saturated brine (30 mL × 3) and then evaporated to dryness under reduced pressure to obtain compound 13-1. Compound 13-1 was not purified and was directly used in the synthesis of compound 13-2. Compound 13-1 was a yellow oil (1.77 g, yield 100%, theoretical value). The mass spectrometry data of compound 13-1 were: ESI-MS (m / z): 1042.52 [M+H] + .

[0220] Step 2: Dissolve compound 13-1 (1.77 g, 1.00 eq) in methanol (20 mL) to obtain a solution; add 5% palladium carbon (0.25 g) to the solution, and react at 25 ° C. under a hydrogen atmosphere for 16 hours to obtain a reaction solution; after thin layer chromatography shows that the reaction is complete, filter the reaction solution and take the filter cake; rinse the filter cake with methanol (5 mL) and take the filtrate; evaporate the filtrate to dryness under reduced pressure to obtain compound 13-2. Compound 13-2 is a yellow oil (1.39 g, yield 100%, theoretical value). The mass spectrometry data of compound 13-2 are: ESI-MS (m / z): 820.51 [M+H] + .

[0221] Step 3: Compound 13-2 (0.6 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution. Triethylamine (0.15 g, 2.0 eq) and N-methoxycarbonylmaleimide (0.14 g, 1.2 eq) were added to the solution, stirred evenly, and then reacted at 45°C for 4 hours to obtain a reaction solution. After the reaction was complete as determined by thin-layer chromatography, the reaction solution was cooled to room temperature (25°C), then the pH was adjusted to 7 with trifluoroacetic acid, and purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from 100:0 to 90:10) to obtain compound 13-3. Compound 13-3 was obtained as a yellow oil (0.6 g, 90% yield). The mass spectrometric data of compound 13-3 were: ESI-MS (m / z): 900.42 [M+H] + .

[0222] Step 4: Compound 13-3 (170 mg, 2.70 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution; 11-2 (55 mg, 1.00 eq), N,N-diisopropylethylamine (32 mg, 3.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (48 mg, 1.30 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution, and the mixture was cooled to 0°C and reacted at 0°C for 1 hour to obtain a reaction solution; after HPLC monitoring of the reaction completion, the reaction solution was first purified by reverse phase preparative chromatography (0.01% trifluoroacetic acid aqueous solution, MeCN, % refers to v / v), and then lyophilized to obtain compound LP-18. Compound LP-18 was obtained as a yellow powdery solid (14 mg, yield 12%). The synthetic route of compound LP-18 is shown in Figure 38 The mass spectrometry results of compound LP-18 are shown in Figure 39 The mass spectrum data of compound LP-18 are: LCMS (ESI) [M+H] + : 1635.49[M+H] + .

[0223] Example 2-19: A linker-payload and its preparation This embodiment provides a linker-cytotoxin LP-19, wherein the linker-cytotoxin LP-19 has the following structure: .

[0224] The preparation method of the linker-cytotoxin LP-19 comprises the following steps: Step 1: Dissolve triethylene glycol monomethyl ether (34.68 g, 1.10 eq) and sodium hydroxide (11.52 g, 1.50 eq) in a mixture of THF / H2O (80 mL / 80 mL) to obtain a solution; slowly add the solution dropwise to a solution of p-toluenesulfonyl chloride (40.00 g, 1.00 eq) in THF (80 mL) cooled to 0°C, and react at 0°C for 3 hours to obtain a reaction solution; after thin-layer chromatography (TLC) indicates the completion of the reaction, separate the reaction solution, extract the aqueous layer with ethyl acetate (50 mL × 3), and take the organic phase; wash the organic phase with saturated brine (40 mL) and evaporate to dryness under reduced pressure to obtain compound 14-1. Compound 14-1 is a colorless oil (58.20 g, 95% yield). The mass spectrometric data of compound 14-1 are: ESI-MS (m / z): 319.12 [M+H] + .

[0225] Step 2: Dissolve diethanolamine (13.32 g, 2.00 eq) in DMF (54 mL) to obtain solution A; dissolve triphenylmethane (16.04 g, 1.00 eq) in DCM (34 mL) to obtain solution B; cool solution A and solution B to 0°C, then dropwise add solution B to solution A, and react at 0°C for 24 hours to obtain a reaction solution; after thin layer chromatography shows the reaction is complete, add ether / water (30 mL / 40 mL) to the reaction solution, beat for 2 hours, and then filter to obtain a filter cake; add chloroform / petroleum ether (96 mL / 132 mL) to the filter cake, beat for 2 hours, and then concentrate to obtain compound 14-2. Compound 14-2 is a yellow oil (14.50 g, yield 72%). The mass spectrum data of compound 14-2 is: ESI-MS (m / z): 348.22 [M+H] + .

[0226] Step 3: Compound 14-2 (2.10 g, 1.00 eq) was dissolved in THF (20 mL) to obtain a solution. 14-1 (7.70 g, 4.00 eq) and sodium hydride (2.70 g, 12.00 eq) were added sequentially to the solution, and the temperature was raised to 65°C. The reaction was continued at 65°C for 16 hours to obtain a reaction solution. After the reaction was completed by thin-layer chromatography, the reaction solution was cooled to room temperature and then purified by preparative silica gel column chromatography (during purification, the volume ratio of petroleum ether and ethyl acetate was increased from petroleum ether:ethyl acetate = 95:5 to petroleum ether:ethyl acetate = 0:100) to obtain compound 14-3. Compound 14-3 was obtained as a yellow oil (1.63 g, yield 42%). The mass spectrometric data of compound 14-3 were: ESI-MS (m / z): 640.25 [M+H] + . 1H NMR (400 MHz, Chloroform-d)δ 7.52 (m, 6H), 7.26 (m, 6H), 7.16 (m, 3H), 3.72-3.48 (m, 28H), 3.39 (s, 6H), 2.57 (t, J = 7.0 Hz, 4H). Step 4: Compound 14-3 (1.73 g, 1.00 eq) was dissolved in methanol (8 mL) to obtain a solution. 5 mL of 5% (w / v, g / mL) aqueous hydrochloric acid was added dropwise to the solution at room temperature, and the reaction was continued at room temperature for 30 minutes to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was obtained. The filtrate was concentrated under reduced pressure, diluted with ether (20 mL), and the pH was adjusted to 8 with saturated sodium carbonate solution. The aqueous layer was evaporated to dryness to obtain a residue. Dichloromethane (40 mL) was added to the residue, and the mixture was stirred for 2 hours. The mixture was then filtered and the dichloromethane was evaporated to dryness to obtain compound 14-4. Compound 14-4 was obtained as a yellow oil (0.83 g, 74% yield). The mass spectrometric data of compound 14-4 were: ESI-MS (m / z): 412.31 [M+H] + .

[0227] Step 5: Compound 4-bromo-2-fluorobenzaldehyde (2.10 g, 1.00 eq) was dissolved in DMF (20 mL) to obtain a solution; compound 14-4 (5.60 g, 1.20 eq) and potassium carbonate (1.70 g, 1.20 eq) were added to the solution, stirred evenly, and then reacted at 105°C for 16 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature, then diluted with water (40 mL), and then extracted with ethyl acetate (30 mL×3), and the organic phase was taken; the organic phase was first washed with saturated brine (20 mL×3), and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane and methanol was increased from dichloromethane: methanol = 100:0 to dichloromethane: methanol = 90:10) to obtain compound 14-5. Compound 14-5 was a yellow oil (2.50 g, yield 38%). The mass spectrum data of compound 14-5 was: ESI-MS (m / z): 580.17 [M+H] + . 1 H NMR (400 MHz, Chloroform- d ) δ 10.29 (s, 1H), 7.65 (m, 1H), 7.44 (m, 1H), 7.25 (m,1H), 3.70-3.48 (m, 28H), 3.45 (t, J= 5.6 Hz, 4H), 3.39 (s, 6H). Step 6: Compound 14-5 (2.50 g, 1.00 eq) was dissolved in dichloromethane (30 mL) to obtain a solution. After cooling the solution to 0°C, benzyl(triphenylphosphine)acetate (2.67 g, 1.50 eq) was added, followed by stirring and reaction at 20°C for 18 hours to obtain a reaction solution. After completion of the reaction by thin-layer chromatography, the reaction solution was concentrated under reduced pressure, redissolved in methanol to the original volume, and purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v). Finally, the solution was lyophilized to obtain compound 14-6. Compound 14-6 was obtained as a yellow powdery solid (2.67 g, yield 87%). The mass spectrometric data of compound 14-6 were: ESI-MS (m / z): 712.18 [M+H] + .

[0228] Step 7: Compound 14-6 (2.50 g, 1.00 eq) was dissolved in toluene (25 mL) to obtain a solution; benzyl carbamate (0.64 g, 1.20 eq), palladium acetate (39 mg, 0.05 eq), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (0.17 g, 0.10 eq) and potassium carbonate (0.58 g, 1.20 eq) were added to the solution in sequence, and the mixture was heated to 100 ° C. under a nitrogen atmosphere and continued to react at 100 ° C. for 4 hours to obtain a reaction solution; after thin layer chromatography showed that the reaction was complete, the reaction solution was first cooled to room temperature (25 ° C), then evaporated to dryness under reduced pressure, and then purified by silica gel column chromatography (during purification, the volume ratio of dichloromethane and methanol was increased from dichloromethane: methanol = 100: 0 to dichloromethane: methanol = 90: 10) to obtain compound 14-7. Compound 14-7 was a yellow oil (2.30 g, yield 44%). The mass spectrum data of compound 14-7 was: ESI-MS (m / z): 783.32 [M+H] + .

[0229] Step 8: Compound 14-7 (2.30 g, 1.00 eq) was dissolved in methanol (20 mL) to obtain a solution. 5% palladium on carbon (0.20 g) was added to the solution, and the mixture was reacted at 25°C under a hydrogen atmosphere for 12 hours to obtain a reaction solution. After the reaction was complete by thin-layer chromatography, the reaction solution was filtered to obtain a filter cake. The filter cake was rinsed with methanol (5 mL) and the filtrate was collected. The filtrate was concentrated under reduced pressure and then purified by preparative silica gel column chromatography (during purification, the volume ratio of dichloromethane to methanol was increased from dichloromethane:methanol = 100:0 to dichloromethane:methanol = 90:10) to obtain compound 14-8. Compound 14-8 was obtained as a yellow oil (0.39 g, yield 23%). The mass spectrometric data of compound 14-8 were: ESI-MS (m / z): 561.27 [M+H] + .

[0230] Step nine: Compound 14-8 (0.05 g, 1.00 eq) was dissolved in dichloromethane (5 mL) to obtain a solution; triethylamine (18 mg, 2.00 eq) and N-methoxycarbonylmaleimide (17 mg, 1.20 eq) were added to the solution, stirred evenly, and then reacted at 45 ° C for 4 hours to obtain a reaction solution; after HPLC monitoring of the reaction completion, the reaction solution was first cooled to room temperature and then evaporated to dryness under reduced pressure to obtain compound 14-9. Compound 14-9 was not purified and was directly used in the synthesis of compound LP-19. Compound 14-9 was a yellow oil (0.04 g, yield 70%). The mass spectrometry data of compound 14-9 was: ESI-MS (m / z): 641.28 [M + H] + .

[0231] Step 10: Compound 14-9 (150 mg, 3.16 eq) was dissolved in N,N-dimethylformamide (3 mL) to obtain a solution. Gly-Gly-Phe-Gly-NH-O-CO-Exatecan (HCl) (65 mg, 1.00 eq), N,N-diisopropylethylamine (24 mg, 2.50 eq), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (42 mg, 1.10 eq), and 1-hydroxybenzotriazole (5 mg, 0.50 eq) were added to the solution. The temperature was then cooled to 0°C and the reaction was continued at 0°C for 1 hour to obtain a reaction solution. After the reaction was completed as monitored by HPLC, the reaction solution was purified by reverse-phase preparative chromatography (0.01% trifluoroacetic acid in water, MeCN, % refers to v / v) and then lyophilized to obtain compound LP-19. Compound LP-19 was obtained as a yellow powdery solid (36 mg, 33% yield). The synthetic route of compound LP-19 is shown in Figure 40 The mass spectrometry results of compound LP-19 are shown in Figure 41The mass spectrum data of compound LP-19 are: LCMS (ESI) [M / 2+H] + : 732.36[M / 2+H] + .

[0232] Example 3-1: An antibody-drug conjugate and its preparation This embodiment provides an antibody-drug conjugate, and the preparation method of the antibody-drug conjugate is: Method 1: Different monoclonal antibodies (purity greater than 95%) obtained in Experimental Example 1 were exchanged into PBS (0.012 M PBS buffer, pH 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using Amicon Ultra 30K ultrafiltration tubes to obtain exchange solutions. An aqueous solution containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) (7.5 eq) was added to the exchange solutions. The mixture was reacted in a 37°C water bath for 1.5 hours to completely open the disulfide bonds between the antibodies, yielding reaction solution A. Reaction solution A was cooled to 25°C in a water bath. Deruxtecan (purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd., CAS No. 1599440-13-7, using an MC-GGFG peptide as the linker) and the compounds from Examples 2-1 to 2-19 (LP-1 to LP-19) were dissolved in DMSO to a 10 mM solution. 12.0 eq of each solution was added to Reaction Solution A, and DMSO was supplemented to a final concentration of 10% (v / v) to obtain a reaction system. The reaction system was reacted in a 25°C water bath for 2 hours to obtain Reaction Solution B. Reaction Solution B was purified using Amicon Ultra 30K ultrafiltration tubes or Protein A 4FF agarose purification resin (purchased from Sangon) to remove unconjugated small molecules, yielding the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR) was determined using RP-HPLC / MS. Purity was determined using SEC-HPLC.

[0233] Method 2: Different monoclonal antibodies (purity greater than 95%) obtained in Experimental Example 1 were exchanged into PBS (0.012 M PBS buffer, pH 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using Amicon Ultra 30K ultrafiltration tubes to obtain exchange solutions. An aqueous solution containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) (2.5 eq) was added to the exchange solutions. The mixture was reacted in a 37°C water bath for 1.5 hours to completely open the disulfide bonds between the antibodies, yielding reaction solution A. Reaction solution A was cooled to 25°C in a water bath. MC-VC-PAB-MMAE (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., using the linker MC-Val-Cit-PAB), MC-VA-PABC-Exatecan (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., using the linker MC-Val-Ala-PAB), and the compounds from Examples 2-1 to 2-19 (LP-1 to LP-19) were dissolved in DMSO to a 10 mM solution. 6.0 eq of each solution was added to reaction solution A, and DMSO was supplemented to a final concentration of 10% (v / v) to obtain a reaction system. The reaction system was reacted in a 25°C water bath for 2 hours to obtain reaction solution B. Reaction solution B was purified using Amicon Ultra 30K ultrafiltration tubes or Protein A 4FF agarose purification resin (purchased from Sangon) to remove unconjugated small molecules, yielding the corresponding ADC, which was stored at -80°C. The antibody-drug conjugation ratio (DAR) was calculated using RP-HPLC / MS. The purity was checked using SEC-HPLC method.

[0234] Method 3: The different monoclonal antibodies (purity greater than 95%) obtained in Experimental Example 1 were exchanged into PBS (0.012 M PBS buffer, pH 7.4; final monoclonal antibody concentration 5.0 mg / mL, 1.0 eq) using Amicon Ultra 30K ultrafiltration tubes to obtain exchange solutions. An aqueous solution containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) (3.4 eq) was added to the exchange solutions, and the mixture was reacted in a 37°C water bath for 1.5 hours to completely break the disulfide bonds between the antibodies, yielding Reaction Solution A. Reaction Solution A was cooled to 25°C in a water bath. Compounds (LP-1 to LP-19) from Examples 2-1 to 2-19 were dissolved in DMSO to prepare 10 mM solutions. Each solution was then added at a 10.0 eq volume to Reaction Solution A. DMSO was then added to a final concentration of 10% (v / v) to obtain a reaction system. The reaction system was reacted in a 25°C water bath for 2 hours to obtain Reaction Solution B. Reaction solution B was purified using Amicon Ultra30K ultrafiltration tubes or Protein A 4FF agarose purification resin (purchased from Sangon) to remove unconjugated small molecules. The corresponding ADC was obtained and stored at -80°C. The antibody-drug conjugate ratio (DAR) was calculated using RP-HPLC / MS. Purity was assessed using SEC-HPLC.

[0235] Among them, the antibody-drug coupling ratio (DAR value) detection method is as follows: The sample was diluted to a concentration of 1 mg / mL with 25 mM ammonium bicarbonate aqueous solution, and 1 μL of N-glycosidase F (PNGase F) was added. The reaction was allowed to react at room temperature for 2 hours. After the reaction was complete, dithiothreitol (DTT) was added to a final concentration of 20 mM. The reaction was allowed to react at room temperature for 1 hour before analysis was performed directly on the instrument. Instrument model: Thermo Fisher Vanquish & QE-HF-X. LC parameters are shown in Table 11. Mass spectrometry conditions were: ESI ion source, spray voltage 3.8 kV, sheath gas (N2) flow rate 40 arb, scan range (m / z) 400-6000; data acquisition was performed in positive ion mode using data-dependent ionization (DDA) mode.

[0236] Purity detection method is as follows: SEC purity analysis - SEC-HPLC (size exclusion chromatography). Instrument model: Agilent 1260 liquid chromatograph. Sample preparation: Dilute the sample to approximately 1 mg / mL with mobile phase, centrifuge at 12,000 rpm for 5 minutes, and sample the supernatant for analysis. Chromatographic conditions are shown in Table 12.

[0237] Table 11 Liquid phase parameters

[0238] Table 12 Chromatographic conditions

[0239] Experimental Example 2: Evaluation of the drugability of antibody-drug conjugates The drugability of the antibody-drug conjugate of Example 3-1 was evaluated (drugability evaluation consisted of measuring the DAR value by RP-HPLC / MS and measuring purity by SEC-HPLC, as described in patent application publication number CN116271079A). The evaluation results are shown in Table 13 (in Table 13, ADC-1 to ADC-12, ADC-13 to ADC-18, ADC-21-8, ADC-22, ADC-23, ADC-24-8, ADC-25, ADC-26, ADC-27-8, and ADC-28 were prepared using Method 1 of Example 3-1; ADC-MMAE, ADC-19, ADC-20, ADC-21-4, ADC-24-4, and ADC-27-4 were prepared using Method 2 of Example 3-1; and ADC-27-6 was prepared using Method 3 of Example 3-1).

[0240] As can be seen from Table 13, at an appropriate drug loading amount, the JFab12b antibody can be normally coupled to Deruxtecan, LP-1, LP-2, LP-3, LP-4, LP-5, LP-6, LP-7, LP-8, LP-9, LP-10, and MC-Vc-PAB-MMAE; the JFab13 antibody can be normally coupled to LP-3 and LP-7; the JFab13b antibody can be normally coupled to LP-7, LP-8, LP-11, LP-12, Deruxtecan, LP-13, LP-14, LP-15, LP-16, LP-17, LP-18, and LP-19; the drug-antibody coupling ratio and aggregation degree obtained by coupling meet the expected requirements; however, the aggregation degree after coupling of JFab12b and JFab13b antibodies with MC-VA-PAB-Exatecan exceeds the acceptable range even at the lowest drug loading amount. It can be seen that except for the MC-VA-PAB-Exatecan coupling, the druggability of other studied cases is feasible.

[0241] Table 13 Evaluation of drugability of antibody-drug conjugates (ADCs)

[0242] Experimental Example 3: Evaluation of in vitro cytotoxic activity of antibody-drug conjugates The in vitro cytotoxic activity of the antibody-drug conjugate described in Example 3-1 was evaluated using NCI-H82, SHP-77, and DMS-53 cells (all purchased from the Shanghai Cell Bank, Chinese Academy of Sciences). The in vitro cytotoxic activity evaluation method was as follows: a cell suspension was prepared in cell culture medium supplemented with 10% (v / v) fetal bovine serum. 135 μL of the cell suspension was added to each well of a 96-well plate. Cells were not plated in the first and twelfth columns, and only 135 μL of culture medium was added. The plates were then incubated at 37°C in 5% (v / v) CO2 for 16 hours. The ADC sample to be tested was prepared in PBS buffer to a working solution (10× concentration) in the first well. Serial dilutions were then performed using PBS buffer at the corresponding dilution multiples. 15 μL of the 10× concentration ADC solution was added to each well, and the plates were incubated at 37°C in 5% CO2 for 5 days. Add 15 μL of CCK-8 solution (APExBIO) to each well, incubate in 5% CO2 at 37°C in the dark for 1–4 hours, read absorbance at 450 nm with a microplate reader, and analyze data using GraphPad Prism 5. See Table 14 for different cell plating densities, initial working solution concentrations (10× concentration), and ADC sample dilution factors. See Table 15 for in vitro cytotoxic activity evaluation results.

[0243] As shown in Table 15, with the exception of ADC-20, which exhibited relatively weak in vitro cytotoxic activity, the remaining ADCs in Example 3-1 all exhibited significant in vitro cytotoxic activity against NCI-H82 cells. Furthermore, their cytotoxicity was not significantly different from that of ADC-1 and ADC-18, which were conjugated to deruxtecan. This indicates that the antibody-drug conjugates in Example 3-1 exhibited strong target cell cytotoxic activity, specifically against NCI-H82 cells expressing delta-like ligand 3 (DLL3).

[0244] As shown in Table 15, the antibody-drug conjugates of Example 3-1 all exhibited significant in vitro cytotoxicity against SHP-77 cells. Furthermore, when conjugated with JFab12b, ADC-8 exhibited a lower IC50 compared to ADC-1 conjugated with deruxtecan. When conjugated with JFab13b, ADC-15, ADC-27-6, and ADC-27-8 exhibited lower IC50s compared to ADC-18 conjugated with deruxtecan. ADC-16, ADC-25, and ADC-27-4 showed no significant differences. The remaining ADCs tested exhibited higher IC50s. Consequently, ADC-1, ADC-8, ADC-9, ADC-15, ADC-16, ADC-18, ADC-25, ADC-27-4, ADC-27-6, and ADC-27-8 exhibited potent cytotoxicity against SHP-77 cells expressing delta-like ligand 3 (DLL3).

[0245] As shown in Table 15, the antibody-drug conjugates of Example 3-1 all exhibited significant in vitro cytotoxicity against DMS-53 cells. Furthermore, when conjugated with JFab13b, ADC-16, ADC-24-4, ADC-24-8, ADC-27-4, ADC-27-6, and ADC-27-8 exhibited lower IC50 values compared to ADC-18 conjugated with deruxtecan. There was no significant difference for ADC-25, while the IC50 values for the remaining ADCs tested were higher. Consequently, ADC-16, ADC-18, ADC-24-4, ADC-24-8, ADC-25, ADC-27-4, ADC-27-6, and ADC-27-8 exhibited potent cytotoxic activity against DMS-53 cells expressing delta-like ligand 3 (DLL3).

[0246] Table 14 Plating density of different cells, concentration of working solution in the first well (10× concentration), and dilution multiple of ADC samples

[0247] Table 15 In vitro killing activity of antibody-drug conjugates (ADCs) (NCI-H82, SHP-77, DMS-53 cells)

[0248] In Table 15, “--” means not detected.

[0249] Experimental Example 4: In vivo efficacy evaluation of antibody-drug conjugates Experiment 1: NCI-H82 tumor-bearing mice 1. Test drugs and materials Blank control group: PBS; ADC-15 (treatment group): low dose 2 mg / kg; high dose 5 mg / kg; ADC-25 (treatment group): dose 5 mg / kg; ADC-27-8 (treatment group): dose 5 mg / kg; JFab13b (naked antibody group): dose 5 mg / kg; Female Balb / c nude mice aged 6 to 8 weeks (purchased from Beijing Weitonglihua Company) were injected subcutaneously on the right back with 100 μL of 50% (v / v) Matrigel containing 5 × 10 6 Human small cell lung cancer cells (NCI-H82) were grown to an average volume of 150 mm. 3 Around 6:00 p.m., the mice were randomly divided into 5 groups according to tumor size and mouse body weight, with 5 animals in each group. The day of group administration was defined as day 0.

[0250] Administration was via the tail vein, once weekly for a total of two doses. The experiment was terminated after 21 days of dosing or when tumor volume reached the ethical limit. Mouse body weight and tumor volume were measured twice weekly, and animal survival was observed. Data were recorded using Excel statistical software and plotted using GraphPad Prism software. Following the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated. Tumor volume (V) was calculated as follows: V = 1 / 2 * L. 长 *L 短 2 ; The calculation formula of tumor inhibition rate (TGI%) is: TGI%=(1-(T i -T0) / (V i -V0))); where T i is the mean tumor volume of the treatment group on the i-th day of administration, T0 is the mean tumor volume of the treatment group on the 0th day of administration, V i is the mean tumor volume of the blank control group on the i-th day of administration, and V0 is the mean tumor volume of the blank control group on the 0th day of administration.

[0251] 2. Data Analysis Data statistics were calculated using Excel 2021. The experimental results are shown in Table 16 and Figure 42-43 shown.

[0252] 3. Conclusion The antibody-drug conjugate of Example 3-1 can significantly reduce tumor volume and has good in vivo anti-tumor activity. At the same time, there is no death in the test mice and no significant weight loss, indicating that the test drug has good safety.

[0253] Experiment 2: SHP-77 tumor-bearing mice 1. Test drugs and materials Blank control group: PBS; ADC-15 (treatment group): low dose 3 mg / kg; medium dose 6 mg / kg; high dose 9 mg / kg; ADC-27-8 (treatment group): 6 mg / kg; Female Balb / c nude mice aged 6 to 8 weeks (purchased from Beijing Weitonglihua Company) were injected subcutaneously on the right back with 100 μL of 1×10 50% (v / v) Matrigel. 7 Human small cell lung cancer cells (SHP-77, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were grown to an average volume of 150 mm. 3 Around 6:00 p.m., the mice were randomly divided into 4 groups according to tumor size and mouse body weight, with 5 animals in each group. The day of group administration was defined as day 0.

[0254] Administration was via the tail vein, once weekly for a total of two doses. The experiment was terminated after 21 days of dosing or when tumor volume reached the ethical limit. Mouse body weight and tumor volume were measured twice weekly, and animal survival was observed. Data were recorded using Excel statistical software and plotted using GraphPad Prism software. Following the experiment, mice were euthanized, and the tumor inhibition rate (TGI%) was calculated. Tumor volume (V) was calculated as follows: V = 1 / 2 * L. 长 *L 短 2 ; The calculation formula of tumor inhibition rate (TGI%) is: TGI%=(1-(T i -T0) / (V i -V0))); where T i is the mean tumor volume of the treatment group on the i-th day of administration, T0 is the mean tumor volume of the treatment group on the 0th day of administration, V i is the mean tumor volume of the blank control group on the i-th day of administration, and V0 is the mean tumor volume of the blank control group on the 0th day of administration.

[0255] 2. Data Analysis Data statistics were calculated using Excel 2021. The experimental results are shown in Table 17 and Figures 44 and 45 shown.

[0256] 3. Conclusion The antibody-drug conjugate of Example 3-1 can significantly reduce tumor volume and has good in vivo anti-tumor activity. At the same time, there is no death in the test mice and no significant weight loss, indicating that the test drug has good safety.

[0257] Table 16 Efficacy of ADC on NCI-H82 xenograft tumors in nude mice

[0258] Table 17 Efficacy of ADC on SHP-77 xenografts in nude mice

[0259] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against delta-like ligand 3, characterized in that The light chain variable region of the monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.3, and the heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO.4, a CDR2 with an amino acid sequence as shown in SEQ ID NO.5, and a CDR3 with an amino acid sequence as shown in SEQ ID NO.

6.

2. The monoclonal antibody according to claim 1, wherein The light chain variable region of the monoclonal antibody comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.20 or SEQ ID NO.32; The heavy chain variable region of the monoclonal antibody includes a polypeptide with an amino acid sequence as shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.

31.

3. The monoclonal antibody according to claim 1 or 2, wherein The light chain of the monoclonal antibody comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.24, SEQ ID NO.27 or SEQ ID NO.30; The heavy chain of the monoclonal antibody includes a polypeptide with an amino acid sequence as shown in SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.28 or SEQ ID NO.

29.

4. A multispecific antibody, characterized in that The multispecific antibody comprises an antigen-binding fragment or a single-chain variable region of the monoclonal antibody according to any one of claims 1 to 3.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 3; or, the nucleic acid molecule encodes the multispecific antibody according to claim 4.

6. A recombinant plasmid, characterized in that The recombinant plasmid carries the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that The host cell is transfected with the recombinant plasmid according to claim 6; or the nucleic acid molecule according to claim 5 is integrated into the genome of the host cell.

8. A method for preparing the monoclonal antibody according to any one of claims 1 to 3, characterized in that: The method comprises: inoculating the host cell according to claim 7 into a cell culture medium for culturing to obtain a culture fluid; and separating and extracting the monoclonal antibody according to any one of claims 1 to 3 from the culture fluid.

9. A drug for treating tumors, characterized in that: The drug contains the monoclonal antibody according to any one of claims 1 to 3; or the drug contains the multispecific antibody according to claim 4.

10. Use of the monoclonal antibody according to any one of claims 1 to 3, the multispecific antibody according to claim 4, the nucleic acid molecule according to claim 5, the recombinant plasmid according to claim 6, the host cell according to claim 7, or the method according to claim 8 in the preparation of a medicament for treating tumors.

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