A linker, an antibody-drug conjugate containing the linker, and preparation method and application thereof

Abstract

The invention relates to an antibody-drug conjugate containing a linker, a drug combination containing the antibody conjugate, the use of the linker, and the use of the antibody conjugate in preparing drugs for preventing and / or treating diseases.

Description

Technical Field

[0001] The present invention relates to a linker for an antibody-drug conjugate, an antibody-drug conjugate prepared from the linker, a pharmaceutical composition containing the antibody-drug conjugate, and uses of the antibody-drug conjugate for treating and / or preventing diseases. Background Art

[0002] Currently, conventional chemotherapy remains the treatment of choice for malignant tumors. Traditional chemotherapy, primarily based on cytotoxic drugs, suffers from common drawbacks such as poor targeting, a narrow safety window, significant toxicity, and drug resistance. The emergence of antibody-drug conjugates (ADCs) has provided a novel therapeutic approach for malignant tumors. ADCs consist of three components: an antibody, a small molecule cytotoxic drug (cytotoxin), and a linker that organically combines the two. ADCs leverage the targeting properties of antibodies to concentrate cytotoxic drugs at the tumor target site, enhancing efficacy and reducing toxicity. The released cytotoxic drug can then further kill surrounding tumor cells through a bystander effect. They combine the high targeting properties of monoclonal antibodies with the high activity of cytotoxic drugs in tumor tissue, resulting in highly effective tumor cell killing with fewer side effects than chemotherapy drugs and superior efficacy compared to traditional antibody-based cancer drugs. The high activity, minimal toxicity, and prolonged duration of action of ADCs offer a novel strategy for the "precision treatment" of tumors.

[0003] Epigenetic dysregulation is often associated with human diseases, particularly cancer. Aberrant epigenetic regulation in cancer includes DNA methylation, histone methylation, histone acetylation, and noncoding RNA and mRNA methylation. Epigenetic modifications can alter gene expression without altering the DNA sequence. Abnormal transcription of oncogenes or tumor suppressor genes mediated by epigenetic enzymes is closely associated with the occurrence, development, and prognosis of tumors. Based on the reversibility of epigenetic mechanisms, small molecule compounds targeting epigenetic regulation have become promising therapeutic drugs. These compounds target epigenetic regulatory enzymes, including histone modifiers (methylation and acetylation), DNA methylases, enzymes that specifically recognize post-translational modifications, chromatin remodeling enzymes, and post-transcriptional regulators (Jin Y, Liu T, Luo H, Liu Y, Liu D. Targeting Epigenetic Regulatory Enzymes for Cancer Therapeutics: Novel Small-Molecule Epidrug Development. Front Oncol. 2022; 12: 848221. Published 2022 Mar 28. doi: 10.3389 / fonc.2022.848221). Epigenetic targeted drugs have demonstrated clinical efficacy in hematological malignancies and therapeutic potential in solid tumors (Jin N, George TL, Otterson GA, et al. Advances in epigenetic therapeutics with focus on solid tumors. Clin Epigenetics. 2021; 13 (1): 83.). Peripheral T-cell lymphoma (PTCL) is a typical epigenetic-related disease with unique sensitivity to histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors (alone or in combination) (Scotto L, Kinahan C, Douglass E, et al. Targeting the T-Cell Lymphoma Epigenome Induces Cell Death, Cancer Testes Antigens, Immune-Modulatory Signaling Pathways. Mol Cancer Ther. 2021; 20(8): 1422-1430).

[0004] Despite clinical efficacy in hematological malignancies, epigenetic drugs have not shown significant efficacy as monotherapy in solid tumors. Recent trials have shown potential for efficacy when used in combination with chemotherapy and hormone therapy. Due to the novelty of their mechanisms and their great therapeutic potential, it is important to reconsider the optimal patient selection, drug regimens, study design, and outcome measures (Juo YY, Gong XJ, Mishra A, et al. Epigenetic therapy for solid tumors: from bench science to clinical trials. Epigenomics. 2015; 7(2): 215-235. doi: 10.2217 / epi.14.73)

[0005] Chidamide (Tucidinostat) is a histone deacetylase (HDAC) inhibitor that has been used in clinical treatment. Studies have found that chidamide treatment of pancreatic cancer cell lines significantly reduced the expression of type I HDACs, Caspase-3, and p21, and increased the expression ratio of Bax / Bcl-2. The results suggest that chidamide may inhibit the proliferation of pancreatic tumor cells by downregulating the expression of type I HDACs and p21, and promote mitochondrial apoptosis pathway-dependent cell apoptosis in a dose-dependent manner (Zhao B, He T. Chidamide, a histone deacetylase inhibitor, functions as a tumor inhibitor by modulating the ratio of Bax / Bcl-2and P21 in pancreatic cancer. Oncol Rep. 2015; 33(1): 304-310. doi: 10.3892 / or.2014.3595).

[0006] A research team found that cedabendine can increase histone acetylation of the PD-L1 gene by activating the transcription factor STAT1. The HDAC gene family is frequently amplified in patients with soft tissue sarcoma. Based on the analysis of drug target gene sets, 8 of 11 patients with liposarcoma (73%) had extensive amplification of the HDAC gene family, and by analyzing the TCGA sarcoma cohort, it was verified that 76.65% (197 / 257) of the cases had amplified the HDAC gene family. Class I HDAC expression is associated with poor prognosis in patients with soft tissue sarcoma, and inhibiting its expression can promote apoptosis and upregulation of programmed cell death ligand 1 (PD-L1). The HDAC class I inhibitor cedabendine can significantly increase the expression of PD-L1 in the tumor microenvironment and increase CD8 +T cell infiltration and reduction of MDSCs. In mouse models, the combination of chidamide and anti-PD-1 antibody significantly promoted tumor regression and improved survival. In addition, chidamide combined with anti-PD-1 antibody Toripalimab is effective in patients with advanced and metastatic sarcoma with tolerable side effects (Que Y, Zhang XL, Liu ZX, et al. Frequent amplification of HDAC genes and efficacy of HDAC inhibitor chidamide and PD-1blockade combination in soft tissue sarcoma. J Immunother Cancer. 2021; 9(2): e001696. doi: 10.1136 / jitc-2020-001696).

[0007] In order to meet more clinically unmet needs for the treatment of solid tumors, it is necessary to develop antibody-drug conjugates based on histone deacetylase inhibitors. Summary of the Invention

[0008] Problems to be solved by the invention

[0009] First, in order to develop diverse stable ADCs to meet various clinical needs, new linker-based stable ADCs still need to be further developed. After continuous efforts, the inventors of this application designed a linker having the structure shown in general formula (I).

[0010] Next, the inventors of the present application provide a conjugate represented by the general formula (II) having excellent anti-tumor effects.

[0011] Solutions for solving problems

[0012] The inventors of the present application have conducted in-depth research and found that the linker represented by the general formula (I) and the conjugate represented by the formula (II) can achieve the desired purpose, thereby completing the present invention.

[0013] That is, the present invention relates to the following linkers and conjugates containing the linkers, or stereoisomers, or tautomers, or pharmaceutically acceptable salts, or deuterated compounds, or solvates thereof.

[0014] The first aspect of the present invention relates to a compound represented by formula I or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a deuterated compound, or a solvate thereof.

[0015]

[0016] in:

[0017] L1 is selected from

[0018]

[0019] Preferably, the carbon end of L1 is connected to the N of succinimide, and the carbonyl end is connected to L2;

[0020] m and t are each independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0021] q is 0, 1, 2, or 3;

[0022] p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0023] X is selected from the group consisting of: CH2, O and NH;

[0024] L2 is absent, or is an amino acid residue, or is a peptide residue consisting of 2-10 amino acid residues;

[0025] Preferably, the amino terminus of L2 is connected to L1, and the carbonyl terminus is connected to L3;

[0026] Preferably, the amino acids include but are not limited to phenylalanine (F), glycine (G), valine (V), lysine (K), serine (S), glutamic acid (E), aspartic acid (N), arginine (R), alanine (A), citrulline, and cysteine ​​(C);

[0027] L3 does not exist or is selected from

[0028] Preferably, the amino acid of L3 is connected to L2, and the carbonyl end or carbon end is connected to D;

[0029] D is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor or a benzamide-based histone deacetylase inhibitor.

[0030] In some embodiments, the L1 is selected from:

[0031] In some embodiments, L1 is selected from:

[0032] In some embodiments, m is selected from the group consisting of: 0, 1, 2, 3, 4, and 5. In some embodiments, m is selected from the group consisting of: 1, 3, 4.

[0033] In some embodiments, t is selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, and 7. In some embodiments, t is selected from the group consisting of: 1, 2, 3, 7.

[0034] In some embodiments, q is selected from 0, 1, and 2.

[0035] In some embodiments, p is selected from 10, 11, 12, 13, 14, 15. In some embodiments, p is selected from 11, 12, 13. In some embodiments, p is 12.

[0036] In some embodiments, X is selected from O and NH.

[0037] In some embodiments, L1 is selected from

[0038]

[0039] In some embodiments, L1 is selected from

[0040]

[0041] In some embodiments, L1 is selected from

[0042]

[0043] In some embodiments, L1 is selected from

[0044]

[0045] In some embodiments, L1 is

[0046] In some embodiments, L1 is

[0047] In some embodiments, L1 is

[0048] In some embodiments, L1 is

[0049] In some embodiments, L2 is absent, or is an amino acid residue, or is a peptide residue consisting of 2-4 amino acid residues.

[0050] In some embodiments, the amino acids include but are not limited to phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline, and cysteine.

[0051] In some embodiments, the amino acids include but are not limited to phenylalanine (F), glycine (G), valine (V), alanine (A), and citrulline.

[0052] In some embodiments, L2 is absent, or is a citrulline residue, or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue, glycine residue-glycine residue-glycine residue, glycine residue-phenylalanine residue-glycine residue, valine residue-cysteine-phenylalanine residue-glycine residue.

[0053] In some embodiments, L2 is absent, or is a citrulline residue, or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue.

[0054] In some embodiments, the L2 is absent or selected from

[0055] In some embodiments, the L2 is absent or selected from

[0056] In some embodiments, L2 is absent or selected from

[0057] In some embodiments, L2 is absent or selected from In some embodiments, L2 is selected from In some embodiments, L2 is In some embodiments, L3 is In some embodiments, D is a benzamide-based histone deacetylase inhibitor.

[0058] In some embodiments, D is selected from

[0059] in,

[0060] A is phenyl or pyridyl, and the phenyl or pyridyl is independently optionally substituted by 1-4 substituents selected from the following: halogen, C1-C4 alkyl (such as methyl, ethyl), C1-C4 haloalkyl (such as trifluoromethyl);

[0061] Preferably, A is phenyl or pyridyl, and the phenyl or pyridyl is independently optionally substituted by 1-2 substituents selected from the following: halogen, C1-C4 alkyl (such as methyl, ethyl), trifluoromethyl;

[0062] More preferably, A is pyridyl, and the pyridyl is optionally substituted with 1-2 substituents selected from the group consisting of: C1-C4 alkyl;

[0063] Further preferably, A is pyridyl;

[0064] Most preferably, A is

[0065] B is phenylene;

[0066] Preferably, B is

[0067] Y is -CO-NH-CH2-;

[0068] Preferably, the methylene end of Y is connected to B, and the carbonyl end is connected to the alkenyl carbon or O;

[0069] R 1 、R 2 Each is independently selected from hydrogen, C1-C4 alkyl;

[0070] Preferably, R 1 、R 2 All are hydrogen;

[0071] X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen, halogen, C1-C4 alkyl, and the remaining three are hydrogen;

[0072] Preferably, X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen and halogen, and the remaining three are hydrogen;

[0073] More preferably, X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen and fluorine, and the remaining three are hydrogen;

[0074] Most preferably, X2 is selected from hydrogen, fluorine, X 1 、X 3 、X 4 For hydrogen.

[0075] In some embodiments, D is selected from

[0076] In some embodiments, D is

[0077] In some embodiments, the compound is selected from:

[0078]

[0079] The second aspect of the present invention relates to the use of the aforementioned compound or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate in the preparation of a ligand drug conjugate (such as an antibody drug conjugate).

[0080] The third aspect of the present invention relates to a ligand-drug conjugate of formula II or its stereoisomers, tautomers, pharmaceutically acceptable salts, deuterated compounds, or solvates.

[0081]

[0082] in:

[0083] Ab is a ligand selected from proteins, antibodies, peptides, enzymes and small molecules;

[0084] n is a number between 0.5 and 8.5; for example, n is a number between 0.8 and 5, a number between 1 and 4, a number between 2 and 6, a number between 3 and 7, a number between 4 and 8, a number between 3.5 and 8.5, a number between 3.5 and 4.5, or a number between 6.5 and 8.5; preferably, n is about 1, 2, 3, 4, 5, 6, 7 or 8; preferably, n is about 2, 3, 4, 5, 6, 7 or 8; preferably, n is about 3, 4, 5, 6, 7 or 8;

[0085] L1 is selected from

[0086] Preferably, the carbon end of L1 is connected to the N of succinimide, and the carbonyl end is connected to L2;

[0087] m and t are each independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0088] q is 0, 1, 2, or 3;

[0089] p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0090] X is selected from the group consisting of: CH2, O and NH;

[0091] L2 is absent, or is an amino acid residue, or is a peptide residue consisting of 2-10 amino acid residues;

[0092] Preferably, the amino terminus of L2 is connected to L1, and the carbonyl terminus is connected to L3;

[0093] Preferably, the amino acids include but are not limited to phenylalanine (F), glycine (G), valine (V), lysine (K), serine (S), glutamic acid (E), aspartic acid (N), arginine (R), alanine (A), citrulline, and cysteine ​​(C);

[0094] L3 does not exist or is selected from

[0095] Preferably, the amino acid of L3 is connected to L2, and the carbonyl end or carbon end is connected to D;

[0096] D is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor or a benzamide-based histone deacetylase inhibitor

[0097] In some embodiments, the L1 is selected from:

[0098] In some embodiments, L1 is selected from:

[0099] In some embodiments, m is selected from the group consisting of: 0, 1, 2, 3, 4, and 5. In some embodiments, m is selected from the group consisting of: 1, 3, 4.

[0100] In some embodiments, t is selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, and 7. In some embodiments, t is selected from the group consisting of: 1, 2, 3, 7.

[0101] In some embodiments, q is selected from 0, 1, and 2.

[0102] In some embodiments, p is selected from 10, 11, 12, 13, 14, 15. In some embodiments, p is selected from 11, 12, 13. In some embodiments, p is 12.

[0103] In some embodiments, X is selected from O and NH.

[0104] In some embodiments, L1 is selected from

[0105] In some embodiments, L1 is selected from

[0106] In some embodiments, L1 is selected from

[0107]

[0108] In some embodiments, L1 is selected from

[0109] In some embodiments, L1 is

[0110] In some embodiments, L1 is

[0111]

[0112] In some embodiments, L1 is In some embodiments, L1 is

[0113] In some embodiments, L2 is absent, or is an amino acid residue, or is a peptide residue consisting of 2-4 amino acid residues.

[0114] In some embodiments, the amino acids include but are not limited to phenylalanine (F), glycine (G), valine (V), alanine (A), citrulline, and cysteine.

[0115] In some embodiments, the amino acids include but are not limited to phenylalanine (F), glycine (G), valine (V), alanine (A), and citrulline.

[0116] In some embodiments, L2 is absent, or is a citrulline residue, or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue, glycine residue-glycine residue-glycine residue, glycine residue-phenylalanine residue-glycine residue, valine residue-cysteine-phenylalanine residue-glycine residue.

[0117] In some embodiments, L2 is absent, or is a citrulline residue, or is selected from the following peptide residues: valine residue-alanine residue, valine residue-citrulline residue, glycine amino-glycine residue-phenylalanine residue-glycine residue, valine residue-alanine residue-phenylalanine residue-glycine residue.

[0118] In some embodiments, the L2 is absent or selected from

[0119] In some embodiments, the L2 is absent or selected from

[0120] In some embodiments, L2 is absent or selected from

[0121] In some embodiments, L2 is absent or selected from

[0122] In some embodiments, L2 is selected from

[0123] In some embodiments, L2 is

[0124] In some embodiments, L3 is

[0125] In some embodiments, D is a benzamide-based histone deacetylase inhibitor.

[0126] In some embodiments, D is selected from

[0127] in,

[0128] A is phenyl or pyridyl, and the phenyl or pyridyl is independently optionally substituted by 1-4 substituents selected from the following: halogen, C1-C4 alkyl (such as methyl, ethyl), C1-C4 haloalkyl (such as trifluoromethyl);

[0129] Preferably, A is phenyl or pyridyl, and the phenyl or pyridyl is independently optionally substituted by 1-2 substituents selected from the following: halogen, C1-C4 alkyl (such as methyl, ethyl), trifluoromethyl;

[0130] More preferably, A is pyridyl, and the pyridyl is optionally substituted with 1-2 substituents selected from the group consisting of: C1-C4 alkyl;

[0131] Further preferably, A is pyridyl;

[0132] Most preferably, A is

[0133] B is phenylene;

[0134] Preferably, B is

[0135] Y is -CO-NH-CH2-;

[0136] Preferably, the methylene end of Y is connected to B, and the carbonyl end is connected to the alkenyl carbon or O;

[0137] R 1 、R 2 Each is independently selected from hydrogen, C1-C4 alkyl;

[0138] Preferably, R 1 、R 2 All are hydrogen;

[0139] X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen, halogen, C1-C4 alkyl, and the remaining three are hydrogen;

[0140] Preferably, X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen and halogen, and the remaining three are hydrogen;

[0141] More preferably, X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen and fluorine, and the remaining three are hydrogen;

[0142] Most preferably, X 2 is selected from hydrogen, fluorine, X 1 、X 3 、X 4 is hydrogen;

[0143] More preferably, D is selected from

[0144] In some embodiments, D is

[0145] In some embodiments, the Ab is an antibody selected from, but not limited to, an anti-EGFR antibody, an anti-CD20 antibody, or an anti-PD-L1 antibody.

[0146] In some embodiments, Ab is selected from, but not limited to, Cetuximab, Panitumumab, Necitumumab, Rituximab, Tositumomab + Iodine 131 Tositumomab, Ofatumumab, Obinutuzumab, Ocrelizumab, Atezolizumab, Avelumab, Durvalumab, and Cemiplimab-rwlc.

[0147] In some embodiments, Ab is Atezolizumab, Avelumab.

[0148] In some embodiments, the ligand drug conjugate is selected from:

[0149]

[0150] In some embodiments, the ligand drug conjugate is selected from:

[0151]

[0152]

[0153] The fourth aspect of the present invention relates to a pharmaceutical composition comprising the aforementioned compound or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate; or comprising the aforementioned ligand-drug conjugate or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate; optionally, further comprising a pharmaceutically acceptable carrier or excipient.

[0154] The fifth aspect of the present invention relates to the use of the aforementioned compound or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate, or the aforementioned ligand-drug conjugate or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate, or the aforementioned pharmaceutical composition in the preparation of a drug for treating or preventing a disease.

[0155] In some embodiments, the disease is a tumor.

[0156] The sixth aspect of the present invention relates to the aforementioned compound or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate, or the aforementioned ligand-drug conjugate or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate, or the aforementioned pharmaceutical composition, which is used to treat or prevent a disease.

[0157] In some embodiments, the disease is a tumor.

[0158] The seventh aspect of the present invention relates to a method for treating or preventing a disease, which comprises administering to a subject an effective amount of the aforementioned compound or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate, or the aforementioned ligand-drug conjugate or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate, or the aforementioned pharmaceutical composition.

[0159] In some embodiments, the disease is a tumor.

[0160] An eighth aspect of the present invention relates to a method for preparing a compound of Formula I or a stereoisomer thereof, a tautomer thereof, a pharmaceutically acceptable salt thereof, a deuterated compound thereof, or a solvate thereof, comprising the steps of:

[0161] The compound represented by formula I-1 is reacted with D' to obtain the compound represented by formula I; or the compound represented by formula I-2 is reacted with H-L2-L3-D to obtain the compound represented by formula I; or the compound represented by formula I-3 is reacted with H-L2-L3-D to obtain the compound represented by formula I;

[0162]

[0163] Preferably, the method comprises the following steps:

[0164] The compound represented by formula I-1 is reacted with D' to obtain the compound represented by formula I; or the compound represented by formula I-2 is reacted with H-L2-L3-D to obtain the compound represented by formula I;

[0165]

[0166] Wherein, LE is a leaving group; preferably, LE is halogen,

[0167] D' is a histone deacetylase inhibitor drug selected from a thiol-based histone deacetylase inhibitor, a hydroxamic acid-based histone deacetylase inhibitor, or a benzamide-based histone deacetylase inhibitor;

[0168] Preferably, D' is a benzamide-based histone deacetylase inhibitor;

[0169] More preferably, D' is selected from

[0170] in,

[0171] A is phenyl or pyridyl, and the phenyl or pyridyl is independently optionally substituted by 1-4 substituents selected from the following: halogen, C1-C4 alkyl (such as methyl, ethyl), C1-C4 haloalkyl (such as trifluoromethyl);

[0172] Preferably, A is phenyl or pyridyl, and the phenyl or pyridyl is independently optionally substituted by 1-2 substituents selected from the following: halogen, C1-C4 alkyl (such as methyl, ethyl), trifluoromethyl;

[0173] More preferably, A is pyridyl, and the pyridyl is optionally substituted with 1-2 substituents selected from the group consisting of: C1-C4 alkyl;

[0174] Further preferably, A is pyridyl;

[0175] Most preferably, A is

[0176] B is phenylene;

[0177] Preferably, B is

[0178] Y is -CO-NH-CH2-;

[0179] Preferably, the methylene end of Y is connected to B, and the carbonyl end is connected to the alkenyl carbon or O;

[0180] R 1 、R 2 Each is independently selected from hydrogen, C1-C4 alkyl;

[0181] Preferably, R 1 、R 2 All are hydrogen;

[0182] X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen, halogen, C1-C4 alkyl, and the remaining three are hydrogen;

[0183] Preferably, X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen and halogen, and the remaining three are hydrogen;

[0184] More preferably, X 1 、X 2 、X 3 、X 4 Among them, any one is selected from hydrogen and fluorine, and the remaining three are hydrogen;

[0185] Most preferably, X 2 is selected from hydrogen, fluorine, X 1 、X 3 、X 4 is hydrogen;

[0186] More preferably, D' is selected from (i.e. Chidamide), (i.e., Entinostat);

[0187] More preferably, D' is (i.e., Chidamide);

[0188] The definitions of L1, L2, L3, and D are as described above.

[0189] The ninth aspect of the present invention relates to a method for preparing a ligand-drug conjugate of Formula II or a stereoisomer thereof, a tautomer thereof, a pharmaceutically acceptable salt thereof, a deuterated compound thereof, or a solvate thereof, comprising the following steps:

[0190] The compound represented by Formula I, or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate reacts with Ab to obtain the ligand-drug conjugate represented by Formula II, or its stereoisomer, or its tautomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its solvate;

[0191] Preferably, the reaction is carried out at pH = 5-10 and temperature of 0-40°C;

[0192]

[0193] in:

[0194] The definitions of Ab, n, L1, L2, L3, and D are as described above.

[0195] In some embodiments, the compound of Formula I is selected from:

[0196]

[0197]

[0198] In some embodiments, the ligand drug conjugate of Formula II is selected from:

[0199]

[0200]

[0201] In some embodiments, the ligand drug conjugate of Formula II is selected from:

[0202]

[0203]

[0204] Beneficial effects of the present invention:

[0205] 1. The present invention provides a new stable ADC linker and an antibody conjugate with excellent anti-tumor effect.

[0206] 2. In the in vitro tumor cell inhibition experiment, compared with naked antibodies, small molecule drugs and combined drugs, the conjugate containing the novel stable linker of the present invention has a significant effect of inhibiting cell growth.

[0207] 3. In the in vivo mouse efficacy experiment, compared with naked antibodies and combined drugs, the conjugate containing the novel stable linker of the present invention has a significant effect in inhibiting tumor growth.

[0208] 4. The conjugate containing the novel stabilizing linker of the present invention has good plasma stability.

[0209] 5. The novel stable linker-containing conjugate of the present invention has excellent endocytosis.

[0210] 6. The novel stable linker-containing conjugate of the present invention has a significant promoting effect on cellular histone acetylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0211] Figure 1-1 This is the RP-HPLC detection chart of ADC-1.1;

[0212] Figure 1-2 This is the RP-HPLC detection chart of ADC-1.2;

[0213] Figure 1-3 This is the RP-HPLC detection chart of ADC-1.3;

[0214] Figure 1-4 This is the RP-HPLC detection chart of ADC-1.4;

[0215] Figure 1-5 This is the RP-HPLC detection chart of ADC-1.5;

[0216] Figure 1-6 This is the RP-HPLC detection chart of ADC-1.6;

[0217] Figure 2 This is the RP-HPLC detection chart of ADC-2;

[0218] Figure 3-1 This is the RP-HPLC detection chart of ADC-3.1;

[0219] Figure 3-2 This is the RP-HPLC detection chart of ADC-3.2;

[0220] Figure 3-3 This is the RP-HPLC detection chart of ADC-3.3;

[0221] Figure 3-4 This is the RP-HPLC detection chart of ADC-3.4;

[0222] Figure 4 This is the RP-HPLC detection chart of ADC-4;

[0223] Figure 5 This is the RP-HPLC detection chart of ADC-5;

[0224] Figure 6 This is the RP-HPLC detection chart of ADC-6;

[0225] Figure 7 RP-HPLC detection chart of ADC-7;

[0226] Figure 8 This is the affinity test result of Ate antibody and ADC-3.3 with hPD-L1-his;

[0227] Figure 9 This is the result of affinity test between Ate antibody and hPD-L1-his;

[0228] Figure 10 This is the result of affinity test between ADC-1.4 and hPD-L1-his;

[0229] Figure 11 This is the result of affinity test between ADC-1.5 and hPD-L1-his;

[0230] Figure 12 This is the result of affinity test between ADC-1.6 and hPD-L1-his;

[0231] Figure 13 This is the result of affinity test between Ate antibody and Cyno PD-L1-his;

[0232] Figure 14 This is the result of affinity test between ADC-1.4 and Cyno PD-L1-his;

[0233] Figure 15 This is the result of affinity test between ADC-1.5 and Cyno PD-L1-his;

[0234] Figure 16 This is the result of affinity test between ADC-1.6 and Cyno PD-L1-his;

[0235] Figure 17 This is the result of affinity test between Ate antibody and mPD-L1-his;

[0236] Figure 18 This is the result of affinity test between ADC-1.4 and mPD-L1-his;

[0237] Figure 19 This is the result of affinity test between ADC-1.5 and mPD-L1-his;

[0238] Figure 20 This is the result of affinity test between ADC-1.6 and mPD-L1-his;

[0239] Figure 21 This is the result of affinity test between Ate antibody and Rat PD-L1-his;

[0240] Figure 22 This is the result of affinity test between ADC-1.4 and Rat PD-L1-his;

[0241] Figure 23 This is the result of affinity test between ADC-1.5 and Rat PD-L1-his;

[0242] Figure 24 This is the result of affinity test between ADC-1.6 and Rat PD-L1-his;

[0243] Figure 25 The figure shows the test results of the effects of ADC-1.4, ADC-1.5, and ADC-1.6 on the acetylation level of histone H4 in NCI-H292 cells;

[0244] Figure 26 This is the result chart of free toxin detection for ADC-1.4, ADC-1.5, ADC-1.6, and ADC-3.3;

[0245] Figure 27 Figure 2 shows the stability test results of ADC-1.6 in human, cynomolgus monkey, SD rat and C57 mouse plasma;

[0246] Figure 28 This is the result of detecting the internalization rate of ADC-1.6 in tumor cells;

[0247] Figure 29 This is the result of ADC-3.3 inhibiting the proliferation of colorectal cancer cell line CT26 hPD-L1-EX cells;

[0248] Figure 30 This is the result of ADC-1.6 inhibiting cell proliferation in the colorectal cancer cell line CT26 hPD-L1-EX;

[0249] Figure 31 Figure 2 is the result of ADC-1.6 inhibiting the proliferation of human malignant melanoma cell line A375 in a co-culture system of human peripheral blood mononuclear cells PBMC and human malignant melanoma cell line A375;

[0250] Figure 32 Figure 2 shows the results of ADC-1.6 inhibiting cell proliferation of human malignant melanoma cell line A375 in a co-culture system of human peripheral blood mononuclear cells (PBMC) and human malignant melanoma cell line A375 (sample concentration: 0.008 μM);

[0251] Figure 33 This is the result of tumor inhibition rate of ADC-1.6 and naked antibody in C57BL / 6J mouse MC38 tumor model;

[0252] Figure 34 The figure shows the tumor inhibition rate of ADC-1.6 and combination in the MC38 tumor model of C57BL / 6J mice. DETAILED DESCRIPTION

[0253] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0254] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. When a brand name is used herein, unless the context indicates otherwise, the brand name includes the product formulation, generic drug, and active ingredient of the brand name product.

[0255] As used herein, "antibody" refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of the heavy chains of immunoglobulins differ, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Ig can be further divided into different subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five classes of Ig can have either kappa or lambda chains.

[0256] The term "drug-to-antibody ratio (DAR)" refers to the average number of cytotoxic drugs (i.e., D in Formula I or Formula II) loaded on each Ab (e.g., antibody) in Formula II (e.g., n in Formula II), which can be an integer or a decimal. The range of DAR (e.g., n in Formula II) can be that each Ab (e.g., antibody) is linked to an average of 0.5 to 8.5 (i.e., any integer or decimal selected from 0.5-8.5, inclusive of the endpoints 0.5 and 8.5) cytotoxic drugs (i.e., D in Formula I or Formula II). For example, DAR (e.g., n in Formula II) can be 0.5, 1, 1.5, 1.94, 2, 2.5, 3, 3.37, 3.5, 4, 4.08, 4.5, 4.71, 4.74, 4.99, 5, 5.26, 5.43, 5.5, 6, 6.34, 6.46, 6.5, 7, 7.08, 7.5, 7.66, 7.77, 7.80, 7.81, 8 or 8.5, etc.

[0257] The term "pharmaceutically acceptable salt" refers to a salt of a compound or conjugate of the present invention that is safe and effective for use in mammals and exhibits the desired biological activity. For example, an acidic group in the compound or conjugate of the present invention may form a salt with a base, non-limiting examples of which include sodium, potassium, calcium, or magnesium salts. Alternatively, a basic group in the compound or conjugate of the present invention may form a salt with an acid, non-limiting examples of which include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0258] The term "solvate" refers to a pharmaceutically acceptable solvate formed between the compound or conjugate of the present invention and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0259] The term "stereoisomer" refers to a compound with the same molecular formula in which the atoms or substituents are connected in the same order but arranged in space in different ways, which is a phenomenon of isomerism in the field of organic chemistry.

[0260] The term "tautomers" refers to two isomers containing heteroatoms (such as nitrogen, oxygen or sulfur atoms), whose structures differ only in the migration of protons and corresponding double bonds, and these two isomers coexist in an equilibrium system and convert into each other at a relatively high rate. A typical example is keto-enol tautomerism.

[0261] The term "deuterated compound" refers to a structure in which one or more hydrogen atoms in the compound or conjugate of the present invention are replaced by deuterium atoms.

[0262] The term "amino acid residue" refers to the incomplete amino acid structure remaining after the amino group of an amino acid loses a hydrogen and the carboxyl group loses a hydroxyl group, and has an amino end and a carbonyl end. In the present invention, L2 is an amino acid residue or a peptide residue composed of 2-10 (preferably 2-4) amino acid residues, wherein the types of the 2-10 (preferably 2-4) amino acids may be the same or different from each other. For example, if L2 is a peptide residue composed of 4 amino acid residues, and the amino acid is selected from glycine and phenylalanine, then L2 can be the following peptide residue: glycine residue-glycine residue-phenylalanine residue-glycine residue (Gly-Gly-Phe-Gly), specifically can be

[0263] In the present invention, unless otherwise explicitly stated, the description method "... are independently selected from" used throughout this document can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, and can also mean that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0264] The substituents of the compounds of the present invention are disclosed by group class or range. It is specifically noted that the present invention includes each independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0265] As used herein, "substituted" or "substituted by" means that any one or more hydrogen atoms on a designated atom or group are replaced by the selection of a designated group, provided that the normal valence of the designated atom is not exceeded.

[0266] In the present invention, "optionally" means that the group may or may not be selected. For example, "A is pyridyl, and the pyridyl is optionally substituted with 1-2 substituents selected from the group consisting of C1-C4 alkyl" means that A is pyridyl, and the pyridyl may or may not be substituted with a substituent.

[0267] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0268] The term "C1-C4 alkyl" refers to an alkyl group having 1 to 4 carbon atoms, preferably a "C1-C3 alkyl group." Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and the like.

[0269] The term "C1-C4 haloalkyl" refers to a group in which one or more hydrogen atoms in any of the above C1-C4 alkyl groups are replaced by halogen (preferably fluorine), examples of which include monofluoromethyl, difluoromethyl, difluoroethyl, trifluoromethyl, and the like.

[0270] The term "absence" indicates that the groups on both sides are directly connected. For example, formula I If L2 does not exist, the structural formula of formula I becomes Accordingly, those skilled in the art will understand that if L2 exists, one end of L2 is connected to L1 and the other end is connected to L3, but if L2 does not exist, L1 is connected to L3. Other similar definitions can be understood with reference to the above content.

[0271] The term "carrier" refers to a system that can change the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also having good membrane permeability, making them excellent drug carriers.

[0272] The term "excipient" refers to an additive other than the main drug in a pharmaceutical preparation, which may also be called an excipient. Excipients include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, beeswax, lanolin, etc.

[0273] The term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) relieving the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0274] The term "subject" includes humans and non-human animals. Exemplary human subjects include humans (referred to as patients) suffering from a disease (e.g., a disease described herein) or normal individuals. The term "non-human animal" herein includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0275] The term "effective amount" refers to an amount effective to achieve the desired therapeutic or preventive effect at the necessary dosage and time. A "therapeutically effective amount" of a compound or conjugate of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the compound or conjugate to elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the compound or conjugate outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive effect at the necessary dosage and time. Typically, but not necessarily, a prophylactic dose is administered to a subject before the onset of disease or in the early stages of the disease, so the prophylactic effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor volume; inhibit (i.e., slow down to a certain extent, preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0276] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples where specific conditions are not specified are generally based on conventional conditions or conditions recommended by the manufacturer.

[0277] Example 1 Preparation of Linker-Toxin (Compound 1)

[0278]

[0279] 1) Preparation of compound 1c:

[0280] To a solution of the compound represented by Formula 1a (805 mg, 1.25 mmol) in N,N-dimethylformamide (12 mL) was added 1-hydroxybenzotriazole (506 mg, 3.7 mmol). The mixture was allowed to react at room temperature for 2 h. Entinotide (1b, 470 mg, 1.25 mmol) was then added and the reaction continued at room temperature for 72 h. Upon completion of the reaction, the reaction solution was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% aqueous formic acid = 0:100% to 50%:50%) to obtain the compound represented by Formula 1c (200 mg). ESI-MS (m / z): 882.3 [M+H] + .

[0281] 2) Preparation of compound 1d:

[0282] Trifluoroacetic acid (2 mL) was slowly added dropwise to a solution of the compound represented by Formula 1c (200 mg, 0.23 mmol) in dichloromethane (10 mL) under an ice bath. The reaction was allowed to proceed for 2 h. The reaction solution was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% aqueous formic acid = 0:100% to 40%:60%) to obtain the compound represented by Formula 1d (80 mg). ESI-MS (m / z): 782.4 [M+H] + .

[0283] 3) Preparation of Compound 1:

[0284] To a solution of the compound represented by Formula 1d (80 mg, 0.23 mmol) in N,N-dimethylformamide (10 mL) was added 1-{15-[(2,5-dioxotrihydro-1H-pyrrol-1-yl)oxy]-15-oxy-3,6,9,12-tetraoxy-1-yl}pyrrole-2,5-dione (1e, 26.5 mg, 0.06 mmol) and stirred at room temperature for 2 h. The reaction mixture was concentrated and purified by reverse-phase column chromatography (acetonitrile / 0.05% formic acid aqueous solution = 0:100% to 50%:50%) to obtain Compound 1 represented by Formula 1 (27.7 mg). ESI-MS (m / z): 1109.5 [M+H] + .

[0285] Example 2 Preparation of Linker-Toxin (Compound 2)

[0286]

[0287] To a 50 mL round-bottom flask, 4-((17S,20S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-15,18-dioxo-20-(3-ureidopropyl)-3,6,9,12-tetraoxa-16,19-diazacycloalkan-21-amino)benzyl(4-nitrophenyl)carbonate (2a, 252 mg, 0.29 mmol) and DMA (2 mL) were added sequentially. The atmosphere was purged with nitrogen three times, and HOBT (116 mg, 0.86 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. Chidamide (2b, 112 mg, 0.29 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 96 hours. The resulting reaction solution was directly purified by reverse phase silica gel column chromatography (eluent: acetonitrile / 0.05% formic acid aqueous solution = 0:100%-35%:65%) to obtain compound 2 (15 mg) represented by formula 2. ESI-MS (m / z): 1123.7 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ9.99(s,1H),9.76(s,1H),9.17(s,1H),8.78–8.75(m,2H),8.56(dd,J=4.8,1.6Hz,1H),8.10(d,J=7.5Hz,1H),8.0 0(dt,J=8.0,1.9Hz,1H),7.93(d,J=8.2Hz,2H),7.85(d,J=8.6Hz,1H),7.62–7.39(m,8H),7.33(d,J=8.6Hz,2H),7.01–6.96(m,2H),6.82( d,J=15.9Hz,1H),5.98(t,J=5.6Hz,1H),5.40(s,2H),5.09(s,2H),4.49(d,J=5.9Hz,2H),4.38(q,J=8.1Hz,1H),4.23(dd,J=8.5,6.8Hz,1 H),3.65–3.41(m,18H),3.04–2.91(m,2H),2.51–2.32(m,2H),1.97–1.94(m,1H),1.70–1.57(m,2H),1.44–1.33(m,2H),0.86–0.82(m,6H).

[0288] Example 3 Preparation of Linker-Toxin (Compound 3)

[0289]

[0290] To a 50 mL round-bottom flask, 4-((S)-2-(S)-2-(6-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propionamido)benzyl(pentafluorophenyl)carbonate (3a, 202 mg, 0.29 mmol) and DMA (2 mL) were added sequentially. The atmosphere was purged with nitrogen three times, and HOBT (116 mg, 0.86 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. Chidamide (2b, 112 mg, 0.29 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 96 hours. The resulting reaction solution was directly purified by reverse-phase silica gel column chromatography (eluent: acetonitrile / 0.05% aqueous formic acid = 0:100% to 35%:65%) to obtain compound 3 (22 mg) of formula 3. ESI-MS (m / z): 903.4 [M+H] + .

[0291] Example 4 Synthesis of Linker-Toxin (Compound 4)

[0292]

[0293] To a 50 mL round-bottom flask, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (4a, 200 mg, 0.27 mmol) and anhydrous N,N-dimethylacetamide (1 mL) were added sequentially. The atmosphere was replaced with nitrogen three times, and HOBT (109 mg, 0.54 mmol) was added thereto. The reaction mixture was stirred at room temperature for 2 hr. Chidamide (2b, 105 mg, 0.27 mmol) was added thereto, and the reaction mixture was stirred at room temperature for 72 hr. The solvent was evaporated under reduced pressure, and the resulting residue was purified by reverse-phase silica gel column chromatography (acetonitrile / 0.05% aqueous formic acid solution = 0:100% to 35%:65%) to give compound 4 (18 mg) of formula 4. ESI-MS (m / z): 989.5 [M+H] + .

[0294] Example 5 Preparation of Linker-Toxin (Compound 5)

[0295]

[0296] Referring to the method in Example 4, entinostat (1b) was used instead of chidamide (2b) in Example 4 to obtain compound 5 (65 mg) of formula 5. ESI-MS (m / z): 975.5 [M+H] + .

[0297] Example 6 Preparation of Linker-Toxin (Compound 6)

[0298]

[0299] Referring to the method in Example 4, 4-((2S,5S)-19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7,17-trioxo-2-(3-ureidopropyl)-10,13-dioxo-3,6,16-triazolamide)benzyl(4-nitrophenyl)carbonate (6a) was used instead of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-3-methylbutyrylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (4a) in Example 4 to obtain compound 6 (55 mg) as shown in Formula 6. ESI-MS (m / z): 1092.5 [M+H]. + .

[0300] Example 7: Preparation of Linker-Toxin (Compound 7)

[0301]

[0302] Preparation of compound 7b:

[0303] Chidamide (2b, 107.6 mg, 0.28 mmol) and 1-hydroxybenzotriazole (18.6 mg, 0.14 mmol) were added to a solution of compound 7a (200 mg, 0.28 mmol) in N,N-dimethylacetamide (0.5 mL). The mixture was reacted at 25°C for 48 hours. After completion of the reaction, the reaction solution was purified by reverse-phase column chromatography (acetonitrile: 0.05% aqueous ammonium bicarbonate solution = 0:100% to 40%:60%) to afford 7b (94 mg). ESI-MS (m / z): 933.4 [M+H] + .

[0304] Preparation of compound 7c:

[0305] To a solution of compound 7b (94 mg) in N,N-dimethylformamide (0.8 mL) was added diethylamine (0.2 mL, 2.76 mmol), and the reaction mixture was allowed to react overnight at 25°C. After completion of the reaction, the reaction mixture was purified by reverse phase column chromatography (acetonitrile: 0.05% aqueous formic acid = 0:100% to 40%:60%) to obtain 7c (51 mg). ESI-MS (m / z): 710.28 [M+H] + .

[0306] Preparation of compound 7:

[0307] To a solution of compound 7c (29 mg, 0.04 mmol) in N,N-dimethylformamide (0.5 mL) were added compound 7d (18 mg, 0.04 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (13 mg, 0.04 mmol), and N,N-diisopropylethylamine (5 mg, 0.04 mmol). The mixture was allowed to react overnight at 25°C. After completion of the reaction, the reaction solution was purified by reverse-phase preparative liquid chromatography (acetonitrile: 0.05% aqueous formic acid = 0:100% to 40%:60%) to obtain compound 7 (15 mg) of formula 7. ESI-MS (m / z): 1213.8 [M+H] + .

[0308] Example 8: Preparation of Linker-Toxin (Compound 8)

[0309]

[0310] To a 5 mL eggplant-shaped flask containing a stirrer, compound 7c (29 mg, 0.04 mmol), 1e (18 mg, 0.04 mmol), and N,N-dimethylacetamide (0.5 mL) were added sequentially. N,N-diisopropylethylamine (6 mg, 0.05 mmol) was added. Stir at room temperature for 3 hours. The resulting reaction solution was purified by reverse-phase column chromatography (eluent: acetonitrile: 0.05% aqueous formic acid = 0:100% to 45%:65%) to obtain compound 8 (20 mg, 0.02 mmol, 54.3%) of formula 8. ESI-MS (m / z): 1037.5 [M+H] + . 1 H NMR(400MHz,DMSO)δ9.96(s,1H),9.79(s,1H),9.21(s,1H),8.82–8.76(m,2H),8.58(dd,J=4.8,1.6Hz,1H),8 .49(s,1H),8.20(d,J=7.2Hz,1H),8.04–7.84(m,4H),7.61–7.44(m,8H),7.35(d,J=8.8Hz,2H),7.03–6.98(m, 2H),6.84(d,J=16Hz,1H),5.11(s,2H),4.51(d,J=6Hz,2H),4.44–4.36(m,1H),4.22(dd,J=8.8,6.8Hz,1H),3 .65–3.44(m,18H),2.49–2.33(m,2H),2.02–1.94(m,1H),1.32(d,J=7.2Hz,3H),0.87(dd,J=15.2,6.8Hz,6H).

[0311] Example 9: Preparation of Linker-Toxin (Compound 9)

[0312]

[0313] Preparation of compound 9b:

[0314] Chidamide (2b, 2545.6 mg, 6.52 mmol) and HOBt (528.6 mg, 3.91 mmol) were added to a solution of compound 9a (5000 mg, 6.52 mmol) in N,N-dimethylacetamide (50 mL). The mixture was reacted at 25°C for 72 hours. After completion of the reaction, the reaction solution was purified by reverse-phase column chromatography (acetonitrile: 0.05% aqueous ammonium bicarbonate solution = 0:100% to 40%:60%) to obtain compound 9b (750 mg). ESI-MS (m / z): 509.7 [M / 2+H]. + .

[0315] Preparation of compound 9c:

[0316] To a solution of compound 9b (550 mg, 0.54 mmol) in N,N-dimethylformamide (5 mL) was added diethylamine (0.5 mL), and the reaction mixture was allowed to react overnight at 25°C. After completion of the reaction, the reaction mixture was purified by reverse-phase preparative liquid chromatography to yield compound 9c. ESI-MS (m / z): 398.91 [M / 2+H] + .

[0317] Preparation of compound 9:

[0318] Compound 9d (25 mg, 0.04 mmol) and N,N-diisopropylethylamine (0.02 mL, 0.11 mmol) were added to a solution of compound 9c (31 mg, 0.04 mmol) in N,N-dimethylacetamide (2 mL). The mixture was allowed to react at 25°C for 18 hours. After completion of the reaction, the reaction solution was purified by reverse-phase column chromatography (acetonitrile: 0.05% aqueous formic acid = 0:100% to 50%:50%) to obtain compound 9 (3 mg) of formula 9. ESI-MS (m / z): 650.33 [M / 2+H]. + .

[0319] Example 10 Preparation of Antibody-Drug Conjugates

[0320] 10.1 Preparation of Anti-PD-L1 Antibody Atezolizumab (Ate Antibody):

[0321] The atezolizumab (DrugBank Accession Number: DB11595) antibody gene was codon-optimized and synthesized by Shanghai Sangon Biotechnology Co., Ltd. to construct a plasmid and obtain the expression plasmid pXC-Atezolizumab.

[0322] The plasmid was transferred into CHO cells using electroporation. After cell screening, culture expression, collection of cell fermentation broth, and affinity chromatography, atezolizumab antibody (abbreviated as Ate antibody or Ate in this application) was obtained.

[0323] Table 1-1 Atezolizumab antibody sequence:

[0324]

[0325] 10.2 Preparation of Anti-PD-L1 Antibody Avelumab (Ave):

[0326] The avelumab (DrugBank Accession Number: DB11945) antibody gene was codon-optimized and synthesized by Shanghai Sangon Biotechnology Co., Ltd. to construct a plasmid and obtain the expression plasmid pXC-Avelumab.

[0327] The plasmid was transferred into CHO cells using electrofection. After cell screening, culture expression, collection of cell fermentation broth, and affinity chromatography, Avelumab antibody (abbreviated as Ave antibody or Ave in this application) was obtained.

[0328] Table 1-2 Avelumab antibody sequence:

[0329]

[0330] 10.3 Antibody-Drug Conjugation Reactions

[0331] Preparation of ADC-1.1:

[0332] Preparation of buffered saline solution:

[0333] Buffer-1: Dissolve 4.65 g of L-histidine in 1 L of ultrapure water and adjust the pH to approximately 5.50 with 0.1 mol / L glacial acetic acid. Sterilize the solution by filtration through a 0.22 μm filter membrane, bottle, and store at 4°C for short-term use.

[0334] Buffer-2: Dissolve 8.96 g of Na2HPO4·12H2O and 3.90 g of NaH2PO4·2H2O in 500 mL of ultrapure water, adjust the pH to 8.0 (±0.05), filter through a 0.22 μm filter membrane for sterilization, bottle, and store at 4°C for a short term until use.

[0335] Replacement of Antibody Buffer: Slowly melt the Ate antibody stock solution at 4°C and replace it with Buffer-1 by ultrafiltration to a final concentration of >10 mg / mL. The concentration was measured by UV spectrophotometer.

[0336] Antibody reduction: Use a pipette to accurately transfer 1 eq of antibody and add a certain amount of Buffer-1 to make the final concentration of the antibody approximately 5 mg / mL. Then add 8 eq of 10 mmol / L TCEP solution, mix well, and keep the mixture at 25°C for 120 min.

[0337] Antibody Conjugation: Accurately pipette the required volume of organic solvent (DMA or DMSO) to 10% of the total volume. Add the small molecule payload (10 eq) of Formula 2 to the organic solvent, mix thoroughly, and then slowly add it to the reduced antibody reaction solution. Continue to slowly stir at 25°C and allow the coupling reaction to proceed for 60 minutes.

[0338] Initial purification of the product: After the termination reaction is completed, the product is replaced into Buffer-1 by ultrafiltration and stored at 4°C for short-term storage and at -80°C for long-term storage until use.

[0339] DAR value determination: DAR values ​​were determined using RP-HPLC. The following conditions were used: PLRP-S column, 8μm, 4.6*150mm; column temperature, 80°C; flow rate, 0.8 mL / min; injection volume, 20 μg; detection wavelength, 214 & 280 nm; gradient elution: 30% B at 0 min, 35% B at 5 min, 45% B at 25 min, 90% B at 26-30 min, and 30% B at 31-40 min, where B was ACN containing 0.1% TFA. Sample preparation: Samples were diluted to 3 mg / mL with the appropriate buffer, and DTT was added to a final concentration of 20 mM. Mix thoroughly and inject directly into the sample for analysis.

[0340] Calculation of average DAR value: ① Calculate the peak area percentages of L0 and L1 respectively (the sum of the peak area percentages of L0 + L1 is 100%) and the peak area percentages of H0, H1, H2, and H3 (the sum of the peak area percentages of H0 + H1 + H2 + H3 is 100%); ② Calculate the weighted percentage of each peak, that is, the peak area percentage * the number of conjugated drugs, such as H3 weighted ratio = H3 peak area percentage * 3; ③ Average DAR value = sum of the weighted percentages of each peak * 2 / 100.

[0341]

[0342] The measured n is 7.08. Figure 1-1 .

[0343] Preparation of ADC-1.2:

[0344] ADC-1.2 was prepared using the same preparation steps and DAR determination methods as ADC-1.1, except that the Ate antibody was replaced with the Ave antibody. During antibody reduction, 4 eq of a 10 mmol / L TCEP solution was added. During antibody conjugation, 6 eq of the small molecule was added, with all other conditions remaining the same.

[0345]

[0346] The measured value of n is 3.37. Figure 1-2 .

[0347] Preparation of ADC-1.3:

[0348] The preparation of ADC-1.3 refers to the preparation steps and DAR value detection method of ADC-1.1, with the only difference being that the Ate antibody is replaced by the Ave antibody.

[0349]

[0350] The measured value of n is 4.74. Figure 1-3 .

[0351] Preparation of ADC-1.4:

[0352] The preparation steps and DAR value detection method of ADC-1.4 were similar to those of ADC-1.1, with the difference that 1.2 eq of TCEP was added during antibody reduction and 4 eq of small molecule load was added during coupling.

[0353]

[0354] The measured n is 1.94. Figure 1-4 .

[0355] Preparation of ADC-1.5:

[0356] The preparation steps and DAR value detection method of ADC-1.5 were similar to those of ADC-1.1, with the difference that 4 eq of TCEP was added during antibody reduction and 6 eq of small molecule loading was added during coupling.

[0357]

[0358] The measured value of n is 4.71. Figure 1-5 .

[0359] Preparation of ADC-1.6:

[0360] The preparation steps and DAR value detection method of ADC-1.6 were similar to those of ADC-1.1, with the difference that 10 eq of TCEP was added during antibody reduction and 14 eq of small molecule load was added during coupling.

[0361]

[0362] The measured value of n is 7.66. Figure 1-6 .

[0363] Preparation of ADC-2:

[0364] The preparation of ADC-2 refers to the preparation method of ADC-1.1 and the DAR value detection method, the only difference is that

[0365] The structure shown in Formula 3 replaces Formula 2.

[0366]

[0367] The measured value of n is 6.34. Figure 2 .

[0368] Preparation of ADC-3.1:

[0369] The preparation of ADC-3.1 refers to the preparation method of ADC-1.1 and the DAR value detection method, the only difference is that the structure shown in Formula 4 replaces Formula 2.

[0370]

[0371] The measured value of n is 6.46. Figure 3-1 .

[0372] Preparation of ADC-3.2:

[0373] The preparation steps and DAR value detection of ADC-3.2 were similar to those of ADC-3.1, with the only difference being that 4 eq of 10 mmol / L TCEP solution was added during antibody reduction and 6 eq of the small molecule was added during antibody conjugation.

[0374]

[0375] The measured value of n is 4.99. Figure 3-2 .

[0376] Preparation of ADC-3.3:

[0377] The preparation steps and DAR value detection method of ADC-3.3 were similar to those of ADC-3.1, with the only difference being that 12 eq of 10 mmol / L TCEP solution was added during antibody reduction and 14 eq of the small molecule was added during antibody conjugation. All other conditions were the same.

[0378]

[0379] The measured n is 7.80. Figure 3-3 .

[0380] Preparation of ADC-3.4:

[0381] The preparation steps and DAR value detection method of ADC-3.4 were similar to those of ADC-3.1, with the only difference being that the Ave antibody replaced the Ate antibody. During the reduction of the antibody, 4 eq of 10 mmol / L TCEP solution was added. During the conjugation of the antibody, 6 eq of the small molecule was added. All other conditions were the same.

[0382]

[0383] The measured value of n is 4.08. Figure 3-4 .

[0384] Preparation of ADC-4:

[0385] The preparation of ADC-4 refers to the preparation steps and DAR value detection method of ADC-1.1, replacing Formula 2 with the structure shown in Formula 5, and the other conditions are the same.

[0386]

[0387] The measured value of n is 5.43. Figure 4 .

[0388] Preparation of ADC-5:

[0389] The preparation steps and DAR value detection method of ADC-5 were referred to those of ADC-1.1, except that the small molecule of formula 7 was used instead of the small molecule of formula 2, 12 eq of TCEP was added during antibody reduction, and 10 eq of the small molecule load was added during coupling.

[0390]

[0391] The measured value of n is 7.77. Figure 5 .

[0392] Preparation of ADC-6:

[0393] The preparation steps and DAR value detection method of ADC-6 were referred to those of ADC-1.1, except that the small molecule of formula 8 was used instead of the small molecule of formula 2, 12 eq of TCEP was added during antibody reduction, and 10 eq of the small molecule load was added during coupling.

[0394]

[0395] The measured value of n is 7.66. Figure 6 .

[0396] Preparation of ADC-7:

[0397] The preparation steps and DAR value detection method of ADC-7 were referred to those of ADC-1.1, except that the small molecule of formula 9 was used instead of the small molecule of formula 2, 12 eq of TCEP was added during antibody reduction, and 10 eq of the small molecule load was added during coupling.

[0398]

[0399] The measured value of n is 7.50. Figure 7 .

[0400] Example 11 Detection of Antibody-Drug Conjugate Aggregates

[0401] The ADC detection instrument was a Thermo Vanquish core, using a BioCore SEC-300 5 μm column (4.6 × 300 mm). The mobile phase was 1X PBS at a flow rate of 0.3 mL / min. The elution was isocratic, the analysis time was 20 min, and the detection wavelength was 280 nm. The results are shown in Table 2.

[0402] Table 2 Comparison of antibody-drug conjugate samples and naked anti-SEC

[0403]

[0404]

[0405] Where: / indicates non-existent or not detected.

[0406] The results showed that the purity of the antibody-drug conjugate samples (such as ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3, ADC-5, ADC-6, ADC-7) was comparable to that of the corresponding naked antibodies.

[0407] Example 12 Affinity determination of antibody-drug conjugate samples

[0408] SPR affinity detection

[0409] Biacore T200 was used to detect the affinity between antibody-drug conjugate samples (such as ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3) / naked antibodies (such as Ate antibodies) and Cyno PD-L1-his, hPD-L1-his, mPD-L1-his, and Rat PD-L1-his.

[0410] The method is as follows: the CM5 chip is activated with N-Hydroxysuccinimide (NHS): 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) = 1v:1v, and then the anti-his antibody is fixed and blocked.

[0411] 1ug / mL hPD-L1-his was captured on the chip surface, and ADC-3.3 and Ate antibodies were diluted 2-fold with 1x PBST, with a total of 9 dilution points, ranging from 25nM to 0.39nM. The program was set up, and the sample was inserted and run. The data was kinetically fitted using BiacoreEvaluation Software according to the 1:1 Binding model. The results are shown in Table 3-1 and Figure 8 .

[0412] Table 3-1 Comparison of affinity between antibody-drug conjugate samples (e.g., ADC-3.3) and Ate antibody

[0413] ligand Analytes ka(1 / Ms) kd(1 / s) KD(M) hPD-L1-His Ate antibody 1.26E+06 <![CDATA[<10 -5 ]]> <![CDATA[<1x10 -12 ]]> hPD-L1-His ADC-3.3 1.02E+06 <![CDATA[<10 -5 ]]> <![CDATA[<1x10 -12 ]]>

[0414] 2ug / mL Cyno PD-L1-his was captured onto the chip surface. Antibody-drug conjugate samples and naked antibody samples were diluted 2-fold with 1x PBST for a total of 7 concentration points, with sample concentrations ranging from 25nM to 0.39nM. The program was set up, and the samples were loaded and run. The data were kinetically fitted using Biacore Evaluation Software according to the 1:1 binding model. Repeat the above steps and capture 2μg / mL hPD-L1-his, 5μg / mL mPD-L1-his, and 5μg / mL Rat PD-L1-his onto the chip surface. Dilute the above four samples twice with 1x PBST. Set 8 concentration points for Ate antibody and ADC-1.4, with a concentration range of 25nM-0.39nM. Set 9 points for ADC-1.5 and ADC-1.6, with a concentration range of 50nM-0.19nM. Set the program, put the samples in and run the program. Use Biacore Evaluation Software software according to the 1:1 Binding model for kinetic fitting. The results are shown in Tables 3-2 and Figures 9 to 24 .

[0415] Table 3-2 Comparison of affinity between antibody-drug conjugate samples and Ate antibody

[0416] ligand Analytes ka(1 / Ms) kd(1 / s) KD(M) hPD-L1-His Ate antibody 2.46E+06 <1.00E-05 <1.00E-12 hPD-L1-His ADC-1.4 1.88E+06 <1.00E-05 <1.00E-12 hPD-L1-His ADC-1.5 1.12E+06 <1.00E-05 <1.00E-12 hPD-L1-His ADC-1.6 1.09E+06 <1.00E-05 <1.00E-12 Cyno PD-L1-His Ate antibody 1.58E+06 1.30E-04 8.26E-11 Cyno PD-L1-His ADC-1.4 1.28E+06 9.49E-05 7.43E-11 Cyno PD-L1-His ADC-1.5 1.00E+06 4.24E-05 4.24E-11 Cyno PD-L1-His ADC-1.6 8.94E+05 1.96E-05 2.19E-11 mPD-L1-His Ate antibody 1.76E+06 1.19E-04 6.74E-11 mPD-L1-His ADC-1.4 1.39E+06 1.09E-04 7.87E-11 mPD-L1-His ADC-1.5 8.10E+05 9.57E-05 1.18E-10 mPD-L1-His ADC-1.6 8.01E+05 8.81E-05 1.10E-10 Rat-PD-L1-His Ate antibody 2.86E+06 9.81E-04 3.43E-10 Rat-PD-L1-His ADC-1.4 2.27E+06 1.00E-03 4.41E-10 Rat-PD-L1-His ADC-1.5 1.26E+06 9.32E-04 7.39E-10 Rat-PD-L1-His ADC-1.6 6.40E+05 7.80E-04 1.22E-09

[0417] The results show that the affinity between the antibody-drug conjugate samples of the present application (such as ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3) and Cyno PD-L1-his, hPD-L1-his, mPD-L1-his, and Rat PD-L1-his is comparable to that of naked antibodies (such as Ate antibodies).

[0418] Example 13 Effect of Antibody-Drug Conjugate Samples on Total Cellular Acetylation Levels

[0419] Detection of the effect of the ADC molecules of the present application (such as ADC-1.4, ADC-1.5, ADC-1.6) on the acetylation level of histone H4 in NCI-H292 cells

[0420] ① Collect NCI-H292 cells and count 20 μL of cells; ② Resuspend the cells to 1×10^5 cells / mL;

[0421] ③ Add 100ul / well to a 48-well plate and incubate at 37°C, 5% CO2 overnight; ④ Dilute the ADC molecule according to the set concentration so that the concentration of chidamide carried by the added ADC molecule is consistent (all 19μM), add 100μL / well to the corresponding well, and incubate at 37°C, 5% CO2 for 48h; ⑤ Aspirate and discard the supernatant, add 100μL / well trypsin digestion, 300μL culture medium to terminate, resuspend the cells in a 1.5mL centrifuge tube, and centrifuge at 300rcf at room temperature for 5min; ⑥ Add 200μL of fixative (4% paraformaldehyde) to each tube, immediately pipette 2-3 times, and incubate at 4°C for 15min; ⑦ Add 1mL PBS to each tube, wash once, centrifuge at 700rcf for 5min, and discard the supernatant; ⑧ Add 50μL of membrane permeabilization solution (1% Triton X-100) to each tube, immediately pipette 2-3 times, and incubate at room temperature for 15min; ⑨ Add Anti-acetyl-Histone H4 Antibody-PE, 0.5 μL / tube, incubate at room temperature in the dark for 1 h; ⑩ Add 1 mL of permeabilization buffer to each tube and wash once, centrifuge at 900 rcf for 5 minutes, and discard the supernatant; Add 100 μL of permeabilization solution to each tube, mix thoroughly by pipetting, and then detect using flow cytometry; The data were processed by Graphpad Prism 9.0, and the results are shown in Figure 25 .

[0422] The results show that the antibody-drug conjugate sample ADC (such as ADC-1.4, ADC-1.5, ADC-1.6) of the present application has a significant promoting effect on the acetylation of histone H4 in NCI-H292 cells.

[0423] Example 14 Free Toxin Detection

[0424] The RP-HPLC method was used to detect free linker-toxin in antibody-drug conjugate samples (such as ADC-1.4, ADC-1.5, ADC-1.6, and ADC-3.3).

[0425] HPLC analysis conditions:

[0426] Instrument: Waters e2695

[0427] Chromatographic column: C18 3.5μm,4.6×150mm column

[0428] Mobile phase A: 0.1% TFA in water

[0429] Mobile phase B: 0.1% TFA in ACN

[0430] Detection wavelength: 254nm

[0431] Flow rate: 0.5 mL / min

[0432] Elution gradient: 0-30 min 10%-80% B, 31-35 min 10% B

[0433] Sample preparation: Take 85 μg of the test sample (such as ADC-1.4, ADC-1.5, ADC-1.6, ADC-3.3), mix the sample with 3 μL DMSO for 5 minutes, then add 60 μL of saturated sodium chloride supernatant (dissolved in 30% methanol and acetonitrile) to the test sample, mix for 10 minutes, centrifuge at 2000 rpm for 2 minutes, and take the supernatant for testing.

[0434] Data analysis: Connexon-toxin concentrations of 0.05μg / mL, 0.1μg / mL, 0.2μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, and 5μg / mL were injected as standards and a standard curve was drawn. If free connexon-toxin was present in the sample, its peak area was substituted into the linear equation to calculate the concentration. Figure 26 .

[0435] The results show that no free linker-toxin was detected in the antibody-drug conjugate sample ADC (such as ADC-1.4, ADC-1.5, ADC-1.6, and ADC-3.3) of the present application.

[0436] Example 15 Plasma stability test.

[0437] Plasma stability test of the antibody-drug conjugate samples (such as ADC-1.6) of the present application in plasma of different species

[0438] The specific operations are as follows:

[0439] Prepare the antibody-drug conjugate sample in sterile PBS to a 1 mg / mL stock solution for immediate use. Prepare the solution in a sterile environment, and sterilize all reagent bottles or containers.

[0440] Gently mix the plasma of various species (human, cynomolgus macaque, SD rat, C57 mouse) at room temperature. Pipette 3600 μL of plasma of each species and add 400 μL of test sample working solution to prepare a sample with a final concentration of 100 μg / mL of test sample. Shake gently, mix well, and set aside.

[0441] The final reaction volume for this incubation experiment was 200 μL, and two replicates were performed at each time point. Samples were collected after incubation at 37°C under sterile conditions for 0, 4, 8, 24, 48, 72, 120, and 168 hours. All operations were performed under sterile conditions and in the dark. Samples at each end point were frozen at -70°C to -90°C.

[0442] After sampling is completed, the samples are detected by RP-HPLC method.

[0443] HPLC analysis conditions:

[0444] Instrument: AB SCIEX TRIPLE QUAD™ 4500 LC / MS

[0445] Chromatographic column: Bridge BEH C18, 2.5 μm, 2.1 × 50 mm, Waters

[0446] Mobile phase A: 0.1% FA 2mmol / L ammonium formate aqueous solution

[0447] Mobile phase B: CAN

[0448] Strong wash solution (SNW): MeOH:ACN:IPA:DMSO=1:1:1:1 (containing 0.5% FA), v / v / v / v Weak wash solution (WNW): MeOH:H2O=1:1, v / v

[0449] Sample processing steps:

[0450] Vortex the sample to mix thoroughly; transfer 20 μL of sample to a 96-well plate according to the plate map; add 20 μL of ACN:H2O=1:1, v / v, to the DB and Carryover samples; add 20 μL of internal standard working solution (1000.000 ng / mL tolbutamide internal standard working solution) to the remaining wells; vortex for 1 minute, and then add 160 μL of ACN:MeOH=1:1 containing 0.1% FA to all wells; seal the film, vortex and mix thoroughly at 1000 rpm for 5 minutes, and centrifuge at 4°C, 4700g for 10 minutes; transfer 100 μL of supernatant to a new 96-well plate according to the plate map; seal the film, and inject and analyze. The calculation results are shown in Table 4 and Figure 27 .

[0451] Table 4 Plasma stability of antibody-drug conjugate samples

[0452]

[0453]

[0454] NA means not detected

[0455] The results show that the Ate antibody did not detect small molecule drugs in the plasma of different species. The antibody-drug conjugate sample ADC of this application (such as ADC-1.6) had a shedding rate of less than 1.0% after incubation at 37°C for 168 hours in the plasma of four species: human, cynomolgus monkey, SD rat, and C57 mouse, indicating overall stability.

[0456] Example 16 Detection of Endocytosis of Antibody-Drug Conjugates (ADCs) in Tumor Cells

[0457] Endocytosis detection of the antibody-drug conjugate sample of the present application (such as ADC-1.6) in the human lung cancer cell line NCI-H292 cells

[0458] ① Collect NCI-H292 cells and take 20 μL to count; ② Resuspend the cells to 6×10^6 cells / mL, and add 100 μL / tube to two 1.5 mL EP tubes; ③ Add the diluted antibody-drug conjugate ADC group and antibody group samples, 100 μL / tube, respectively, to a final concentration of 150 nM, mix well, and incubate at 4°C in the dark for 1 hour; ④ Add pre-cooled PBS to wash twice, centrifuge at 1200 rpm at 4°C for 5 minutes, and discard the supernatant; ⑤ Add 300 μL / tube of complete culture medium to resuspend the cells, aspirate 150 μL of cell suspension and place it into a new EP tube, one tube continues to be incubated at 4°C and the other tube at 37°C; ⑥ Incubate at 2 After 1 h, 4 h, and 22 h, 50 μL of cell suspension was taken from the 4°C and 37°C tubes, respectively, and placed in new EP tubes. 1 mL of pre-cooled PBS was added to each tube, and the cells were centrifuged at 4°C, 1200 rpm for 5 min, and the supernatant was discarded. ⑦ 0.5 μL / tube of secondary antibody was added, mixed, and incubated at 4°C in the dark for 30 min. ⑧ Washed once with pre-cooled PBS, centrifuged at 4°C, 1200 rpm for 5 min, and the supernatant was discarded. ⑨ 100 μL / tube of pre-cooled PBS was added, the cells were resuspended, and then tested on the instrument. ⑩ The internalization rate was calculated using the following formula: Internalization (%) = Positive rate 4°C (%) - Positive rate 37 ℃ (%), where the positive rate 4℃ (%) is the positive rate of cells incubated at 4℃, 37 ℃ (%) is the positive rate of cells incubated at 37℃. The data were processed by Graphpad Prism 9.0. Figure 28 .

[0459] The results show that the tumor cell endocytosis rate of the antibody-drug conjugate ADC group (such as ADC-1.6) and the antibody group (such as Ate antibody) increased over time, and the tumor cell endocytosis rate of the antibody-drug conjugate ADC group was significantly better than that of the antibody group.

[0460] Example 17 Inhibitory Effects of Antibody-Drug Conjugate Samples on Tumor Cells

[0461] 17.1 Detection of the Inhibition of CT26-hPD-L1 Cell Proliferation by the ADC Molecules of the Present Application (e.g., ADC-3.3)

[0462] ① Collect CT26 hPD-L1-EX cells and count 20 μL of them; ② Resuspend the cells to 0.5×10^5 cells / mL and 0.5×10^5 cells / mL in 5 mL each; ③ Add 50 μL / well to two 96-well plates and incubate at 37°C, 5% CO2 overnight; ④ Dilute the ADC sample according to the set concentration and add 100 μL / well to the plate; ⑤ Incubate at 37°C, 5% CO2 for 48 hours; ⑥ Aspirate and discard 50 μL of culture medium, then add MTS reagent at 20 μL / well and continue incubating at 37°C, 5% CO2 for 1-4 hours; ⑦ Read at 490 nm. Results are shown in the figure. Figure 29 and Table 5.

[0463] Table 5 Inhibition of CT26 cells by antibody-drug conjugate sample ADC-3.3

[0464]

[0465] The results show that the antibody-drug conjugate sample ADC of the present application (such as ADC-3.3) has a significant inhibitory effect on the proliferation of CT26-hPD-L1 cells, and its inhibitory effect is significantly better than that of a single small molecule drug (such as Chidamide, Chidamide), a single naked antibody (such as Ate antibody), and the combined effect of a small molecule drug and a naked antibody (such as Chidamide + Ate antibody, i.e. Chi + Ate).

[0466] 17.2 Detection of the proliferation inhibition of the ADC molecules of the present application (such as ADC-1.6) on the colorectal cancer cell line CT26

[0467] ① Collect CT26 hPD-L1-EX cells and count 20 μL of them; ② Resuspend the cells to 2 × 10^5 cells / mL; ③ Inoculate 50 μL / well into a 96-well plate (1 × 10^4 cells / well) and culture overnight at 37°C, 5% CO2; ④ The next day, remove the 96-well plate, dilute the sample with complete medium containing 10% FBS, and add 50 μL / well to the plate to a final concentration of 10 μM, which is recorded as Day 0. Continue to culture at 37°C, 5% CO2 for 2 days; ⑤ Day 2: After observing the cell growth status under a microscope, add CCK8 at 10 μL / well and continue to culture at 37°C, 5% CO2 for 4 hours; ⑥ Read the absorbance value (OD) at 450 nm on a microplate reader in real time 450 ), the results are shown in Figure 30 and Table 6.

[0468] Table 6 Inhibition of CT26 cells by antibody-drug conjugate sample ADC-1.6

[0469]

[0470]

[0471] The results show that the antibody-drug conjugate ADC group (such as ADC-1.6) has a greater inhibitory effect on the proliferation of CT26 hPD-L1-EX cells than the small molecule drug group (such as Chidamide), the antibody group (such as Ate antibody) and the combination group (such as Chidamide + Ate antibody, i.e. Chi + Ate).

[0472] 17.3 Detection of the inhibitory effect of the ADC molecules of the present application (such as ADC-1.6) on the human malignant melanoma cell line A375 in the co-culture system of human peripheral blood mononuclear cells PBMC and human malignant melanoma cell line A375

[0473] The specific operations are as follows:

[0474] Remove the frozen PBMC from the liquid nitrogen tank, add 8 mL of preheated RPMI 1640 medium, and centrifuge at 500 g for 10 minutes; discard the supernatant, resuspend the PBMC with a small amount of RPMI 1640 medium, dilute 10 times, and take 20 μL to count; resuspend the cells to 5E6 cells / mL; collect the A375-hPDL1-Luciferase cell suspension, take 20 μL to count; resuspend the cells to 1E6 cells / mL; mix the PBMC and A375-hPDL1-Luciferase cell suspensions in equal volumes to prepare a cell mixture with an E:T ratio of 5:1, and fill it up to 1500 μL with RPMI1640 complete medium; put the matrigel on ice to melt, take 1500 μL of matrigel and mix with the cell mixture, and add 20 μL / well to the black 384-well plate The plate was placed at 37°C for 10 minutes to solidify; ADC-1.6 and Ate antibodies were diluted to 5μM, 1μM, 0.2μM, 0.04μM, 0.008μM, 0.0016μM, and 0μM dilutions, and Chidamide was diluted to 200μM, 40μM, 8μM, 1.6μM, 0.32μM, 0.064μM, 0.0128μM, and 0μM dilutions; 8 concentration gradients of each sample were repeated 3 times, and 30μL / well was added to a 384-well plate, and incubated at 37°C, 5% CO2 for 6 days; the 384-well plate was removed, and 20μL / well of the Bio-LiteLuciferase Assay System was added, incubated for 5-10 minutes, and detected using a multifunctional microplate reader. The results are shown in FIG. Figure 31 、 Figure 32 and Table 7.

[0475] Table 7 Inhibition of A375 cells by antibody-drug conjugate sample ADC-1.6

[0476] Ate antibody ADC-1.6 Combo(Chidamide:Ate antibody=8:1) Chidamide IC50 (μM) 1.09E+08 ＜0.01 0.03206 0.8232

[0477] The results showed that the IC50 values ​​for the antibody-drug conjugate ADC (e.g., ADC-1.6) were less than 0.01 μM, while the IC50 values ​​for the Ate antibody, Chidamide, and combination (Chidamide:Ate antibody = 8:1) groups were all greater than 0.03 μM. Antibody-drug conjugate ADCs (e.g., ADC-1.6) exhibited significant inhibitory effects on A375-hPDL1-Luciferase cell proliferation at lower concentrations compared to small molecule drugs (e.g., Chidamide), antibodies (e.g., Ate), and combinations (e.g., Chidamide + Ate, i.e., Chi+Ate). At a concentration of 0.008 μM, the inhibitory effect of the antibody-drug conjugate ADC (e.g., ADC-1.6) on A375-hPDL1-Luciferase cell proliferation was significantly greater than that of the small molecule drugs (e.g., Chidamide), antibodies (e.g., Ate), and combinations (e.g., Chi+Ate).

[0478] Example 18 Antitumor efficacy of antibody-drug conjugate sample ADC

[0479] In vivo pharmacodynamics studies of the present antibody-drug conjugate ADC (e.g., ADC-1.6) using the C57BL / 6J mouse MC38 tumor model

[0480] 18.1 The specific method is as follows: On the day of cell inoculation, 3×10 5 MC38 cells (0.1 mL / mouse). When the average tumor size of mice is about 70-80 mm 3 The mice were divided into 3 groups at the beginning of the study period. The drug administration was started on the day of grouping. The administration cycle was three times a week, and the administration method was intraperitoneal injection. The naked antibody group and the drug conjugate sample ADC group were intraperitoneally injected with 10 mg / kg Ate antibody and ADC-1.6, respectively. The vehicle control group (Vehicle) was intraperitoneally injected with an equal volume of PBS solution. After the start of drug administration, body weight and tumor volume were measured three times a week. The tumor volume calculation method was: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experiment was terminated on the 7th day after administration, all mice were euthanized, and tumor tissues were taken and weighed. Figure 33 .

[0481] The results show that the tumor volume of the antibody-drug conjugate sample ADC-1.6 of the present application was 455.74 mm on the 7th day after administration. 3 The tumor volumes of the naked antibody group and the vehicle control group were 701.09 mm 3 、821.79mm 3 The tumor inhibition rate TGI (%) of the antibody-drug conjugate sample ADC-1.6 group was 48.97%, which was significantly different from that of the solvent control group (P<0.05), and its inhibitory effect was significantly better than that of the Ate antibody group (TGI=16.26%).

[0482] 18.2 The specific method is as follows: On the day of cell inoculation, 3×10 5 MC38 cells (0.1 mL / mouse). When the average tumor size of mice is about 50-60 mm 3 The mice were divided into 3 groups at the beginning of the study period. The drugs were administered three times a week by intraperitoneal injection. The combination group (Ate antibody + Chidamide) received an intraperitoneal injection of 10 mg / kg mAb and 0.2 mg / kg Chidamide. The drug-coupled sample ADC group received an intraperitoneal injection of 10 mg / kg ADC-1.6. The vehicle control group (Vehicle) received an intraperitoneal injection of an equal volume of PBS solution. After the start of drug administration, body weight and tumor volume were measured three times a week. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experiment was terminated on the 7th day after administration, all mice were euthanized, and tumor tissues were taken and weighed. Figure 34 .

[0483] The results show that the tumor volume of the antibody-drug conjugate sample ADC-1.6 of the present application was 329.63 mm on the 7th day after administration. 3 The tumor volumes of the combination group and the vehicle control group were 457.35 mm 3 、499.89mm 3 The TGI (%) of the antibody-drug conjugate sample ADC-1.6 was 38.26%, which was significantly different from that of the vehicle control group (P<0.05), and its inhibitory effect was significantly better than that of the combination group (TGI=9.63%).

[0484] The present invention has been illustrated through various specific embodiments. However, those skilled in the art will appreciate that the present invention is not limited to these specific embodiments. Those skilled in the art may make various modifications and variations within the scope of the present invention, and the various technical features described throughout this specification may be combined without departing from the spirit and scope of the present invention. Such modifications and variations are within the scope of the present invention.

Claims

1. A ligand-drug conjugate, wherein the ligand-drug conjugate is selected from the following: in: Ab is the anti-PD-L1 antibody Atezolizumab; n is a number between 0.5 and 8.

5.

2. A pharmaceutical composition comprising at least one ligand-drug conjugate according to claim 1 and a pharmaceutically acceptable excipient.

3. Use of the ligand-drug conjugate according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating or preventing a disease, wherein the disease is colorectal cancer or human malignant melanoma.

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