Linker, antibody-drug conjugate, and method for preparing the same

Abstract

The present invention provides a linker, an antibody-drug conjugate, and a method for preparing the same. The drug linker of the present invention has good solubility, can significantly improve the water solubility of existing low-molecular-weight compounds, and improves the stability and uniformity of the conjugate after coupling with an antibody.

Description

[Technical Field]

[0001] This application relates to the field of biopharmaceuticals, and more specifically to linkers, antibody-drug conjugates, and methods for preparing them. [Background technology]

[0002] Antibody-drug conjugates (ADCs) achieve the goal of precisely killing tumors by utilizing the characteristic of monoclonal antibodies to specifically recognize certain antigens on the surface of tumor cells, thereby accurately delivering and releasing antitumor drugs (e.g., small molecule chemotherapeutic drugs) to tumor target cells. Due to their appropriate molecular weight, high stability, strong targeting, and low toxic side effects, ADCs are considered the most promising antitumor drugs. However, there are many challenges that must be considered and resolved in order to successfully develop ADCs. For example, the antibody must specifically recognize the lesion site, have low immunosensitization, and undergo efficient and rapid endocytosis; the antibody-drug linker must have high stability in the blood and specifically activate and efficiently release the small molecule drug within the target cell; and the coupled small molecule drug must have potent cytotoxic activity. Nanobody-drug conjugates (NDCs) prepared using nanobodies offer advantages over conventional ADCs, including high vascular permeability, excellent blood-brain barrier permeability, potent tumor penetration, and rapid delivery to target cells. This enhances drug accumulation in tumors while allowing for appropriate control of drug plasma exposure and half-life. As a result, they are expected to contribute to further improvements in the therapeutic effect against solid tumors and the overall therapeutic range, making them the most promising new antitumor drugs.

[0003] When conventional linkers are used, phenomena such as ADC instability, aggregation, and sedimentation may occur. Furthermore, if the lipophilicity is excessively high, it can lead to rapid metabolism in metabolic organs such as the liver, reducing the pharmacokinetic (PK) properties. Therefore, it is necessary to explore linker structures to optimize the physicochemical properties and pharmacokinetic (PK) properties of ADCs while ensuring a high drug load.

[0004] Therefore, in order to further develop ADC drugs with superior therapeutic effects, the development of novel linkers, antibody-drug conjugates, and methods for preparing them remains necessary in this field. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a novel linker, an antibody-drug conjugate, and a method for preparing the same. [Means for solving the problem]

[0006] A first aspect of the present invention provides a compound or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound has a structure as shown in formula A, [ka] In the formula, Q is a linker group for binding to the antibody. Z1 contains a glucose group containing Y hydroxyl groups, s is an integer between 0 and 10. n is an integer between 1 and 24. r is an integer between 0 and 10. X is a linking group, P1 is a polypeptide residue, P2 is a chemical bond or an AA-PAB structure, where AA is a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment in which 2 to 4 amino acids are linked by peptide bonds), and PAB is a p-aminobenzylcarbamoyl group. D is a drug.

[0007] In another preferred example, the glucose group includes both cyclic and open-chain structures.

[0008] In another preferred example, the glucose group includes its precursor or derivative.

[0009] In another preferred example, in Z1, Y is a positive integer.

[0010] In another preferred example, Y is 4 to 50, preferably 5 to 30, and more preferably 6 to 25.

[0011] In another preferred example, in Z1, Y ≥ 5, preferably ≥ 8.

[0012] In another preferred example, Z1 further comprises an amino group.

[0013] In another preferred example, the structure of Z1 is: [ka] It is selected from the group consisting of the following.

[0014] In another preferred example, the structure of Z1 is: [ka] It is selected from the group consisting of the following.

[0015] In another preferred example, the linker base Q is selected from the following: [ka] Here, A represents an optionally substituted C3 - C8 alkylene group, C3 - C8 alkenyl group, C3 - C8 alkynyl group, C3 - C6 cycloalkenyl group, C3 - C8 cycloalkyl group, or an optionally substituted diethylene glycol - octaethylene glycol acyl group; Ar represents an optionally substituted C5 - 6 aryl group or heteroaryl group; and "*" represents that -C=O- forms an amide bond with an amino group.

[0016] In another preferred example, the above-mentioned optional substitution refers to the substitution at any available linking point of the aryl group.

[0017] In another preferred example, the P1 is , , , C=O , , C=O , , , C=O ,

[0018] , C=O , ,

[0020] , NH , , NH ,

[0019] , NH ,

[0021] , , , , NH , -Val-Cit- C=O , NH -Val-Ala- C=O , NH -Ala-Ala-Ala- C=O , NH -Ala-Ala- C=O , NH -Gly-Gly-Phe-Gly- C=O , NH -Val-Lys- C=O selected from the group consisting of

[0018] In another preferred example, the X is

Chemical formula

[0019] In another preferred example, in the X, 1 and 2 each represent a linking site. For example, 1 represents the linking site to the upper half of formula A, and 2 represents the linking site to the lower half of formula A.

[0020] In another preferred example, the D is a cytotoxic small molecule drug selected from the group consisting of a STING agonist, a KRAS - G12D inhibitor, a tubulin inhibitor, a topoisomerase inhibitor, and a DNA binder.

[0021] In another preferred example, the STING agonist is selected from the group consisting of diABZI analogs.

[0022] In another preferred example, the KRAS-G12D inhibitor is selected from MRTX1133 analogs.

[0023] In another preferred example, the tubulin inhibitor is selected from the group consisting of maytansine derivatives, monomethyl auristatin-E (MMAE), monomethyl auristatin-F (MMAF), monomethyl dolastatin 10 (MMAD), tubulysin derivatives, cryptophycin derivatives, and taltobulin, and is preferably MMAE or MMAF.

[0024] In another preferred example, the topoisomerase inhibitor is selected from the group consisting of SN38, DXd, a derivative of the doxorubicin metabolite PNU-159682, exatecan (DX8951), and a derivative of the irinotecan (CPT-11) metabolite SN38, and is preferably a topoisomerase 1 (Topo1) inhibitor such as SN38, DXd, or exatecan.

[0025] In another preferred example, the DNA binding agent is selected from the group consisting of PBD derivatives and duocarmycin derivatives.

[0026] In another preferred example, the structure of formula A is selected from the group consisting of the following: [ka] TIFF2026519886000008.tif212169TIFF2026519886000009.tif106169

[0027] A second aspect of the present invention provides an antibody-drug conjugate (ADC) which is formed by coupling a compound of formula A described in the first aspect of the present invention with an antibody. In another preferred example, the composite is as shown in formula B, [ka] Here, Ab is an antibody, L is a linker, D is a drug, n is an integer or decimal number between 1 and 10.

[0028] In another preferred example, the antibody includes antigen-binding fragments, nanobodies, chimeric antibodies, bivalent antibodies, and / or polyvalent antibodies.

[0029] In another preferred example, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody, or a chimeric antigen receptor antibody (CAR).

[0030] In another preferred example, the CDR region of the humanized antibody comprises one, two, or three amino acid changes.

[0031] In another preferred example, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0032] In another preferred example, the antibody is a double-chain antibody or a single-chain antibody.

[0033] In another preferred example, the antibody is a nanobody or monoclonal antibody.

[0034] In another preferred example, the antibody is a partially or fully humanized monoclonal antibody.

[0035] In another preferred example, the antibody or nanobody or its fusion protein targets a target selected from the group consisting of TF, EGFR, HER2, HER3, BCMA, B7-H3, CD73, AXL, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H6, GPC3, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, Trop2, TIGIT, LAG-3, TLR7, or a combination thereof.

[0036] In another preferred example, the antigen-binding fragment comprises (i) a Fab fragment, (ii) an F(ab')2 fragment, (iii) an Fd fragment, (iv) an Fv fragment, (v) a single-stranded Fv(scFv) molecule, and (vi) an dAb fragment.

[0037] In another preferred example, the antibody is an antibody or nanobody that targets TF and / or HER2 (an anti-TF and / or HER2 antibody, an anti-TF and / or HER2 nanobody, or a fusion protein thereof).

[0038] In another preferred example, the antigen-binding fragment targeting the TF nanobody has CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.3.

[0039] In another preferred example, the TF target nanobody has a heavy chain variable region as shown in SEQ ID NO. 4.

[0040] In another preferred example, the immune complex comprises a polyvalent (e.g., bivalent) nanobody targeting the TF and / or HER2, or a polyvalent (e.g., bivalent) antibody targeting the TF and / or HER2.

[0041] In another preferred example, the polyvalent nature means that the amino acid sequence of the immune complex contains multiple repeating nanobodies or antibodies that target the TF and / or HER2.

[0042] In another preferred example, the antibody-drug conjugate (ADC) is a monomer, dimer, or polymer.

[0043] In another preferred example, the antibody includes functional domains that can improve the physicochemical properties or drug potential of the protein, such as an Fc segment, an anti-albumin nanobody (HLE), or an albumin-binding domain (ABD).

[0044] In another preferred example, the TF target nanobody comprises an Fc segment, preferably as shown in SEQ ID NO. 5.

[0045] In another preferred example, the HER2-targeted nanobody comprises an Fc segment, preferably as shown in any of SEQ ID NO. 7-9.

[0046] In another preferred example, the antibody-drug conjugate comprises the structure shown in formula (B), [ka] Here, Q is a linker group that can be coupled to an antibody, Z1 is a hydrophilic group containing a hydroxyl group and an amino group-containing glucose group, X is a linking group, P1 is a polypeptide residue, P2 is a directly bound or para-aminobenzoic acid ester (PABC) group, D is an antitumor drug, n is an integer from 1 to 24, Ab is an antibody or nanobody fusion protein, and m = 1 to 8.

[0047] In another preferred example, the antibody-drug conjugate (ADC) is selected from the group consisting of the following: The structure of the composite ADC is as follows: [ka] TIFF2026519886000013.tif213169TIFF2026519886000014.tif106169 Here, Ab is the ligand, and m = 1 to 8.

[0048] A third aspect of the present invention provides a pharmaceutical composition comprising (a) an antibody-drug conjugate described in the second aspect of the present invention or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

[0049] A fourth aspect of the present invention provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the conjugate or a pharmaceutically acceptable salt thereof, in the preparation of an antitumor drug or cancer therapeutic agent.

[0050] In another preferred example, the cancer is selected from the group consisting of lung cancer, liver cancer, breast cancer (triple-negative breast cancer), ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma.

[0051] A fifth aspect of the present invention is: (1) The step of reacting the antibody with a reducing reagent in a buffer to obtain the reduced antibody, (2) A method for preparing an antibody-drug conjugate according to a second aspect of the present invention is provided, comprising the step of crosslinking (coupling) a compound shown in formula A and the reduced antibody obtained in step (1) in a mixture of a buffer and an organic solvent to obtain an antibody-drug conjugate B. [Effects of the Invention]

[0052] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0053] [Figure 1A] This study describes the cytokine induction effects of 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC1-39, and HuSC1-39-L1-AN014 after 48 hours of co-culture treatment in MDA-MB-231 / PBMC. 1000 nM diABZI was used as the positive control, and 150 μg / mL hIgG1 was used as the negative control. Figure 1A shows the detection results for the induction of the chemokine CXCL10. [Figure 1B] This study describes the cytokine induction effects of 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC1-39, and HuSC1-39-L1-AN014 after 48 hours of co-culture treatment in MDA-MB-231 / PBMC. 1000 nM diABZI was used as the positive control, and 150 μg / mL hIgG1 was used as the negative control. Figure 1B shows the detection results for induction of the inflammatory cytokine interferon-γ. [Figure 2A] The experimental results for 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC1-39, and HuSC1-39-L1-AN014 after 48 hours of co-culture treatment in BxPC3-Luc / PBMC are shown. Figure 2A shows the viability of BxPC3-Luc cells detected, where the starting concentration for co-dilution of low molecular weight loading AN014 was 1000 nM, and 150 μg / mL of hIgG1 was used as a negative control. [Figure 2B] The experimental results for 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC1-39, and HuSC1-39-L1-AN014 after 48 hours of co-culture treatment in BxPC3-Luc / PBMC are shown. Figure 2B shows the detection of drug effects that promote the expression and secretion of the inflammatory cytokine Interferon-γ. [Figure 3] The viability of 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC1-39, and HuSC1-39-L1-AN014 cells detected after 24 hours of co-culture treatment in HCC1806-Luc / PBMC was shown. Here, the starting concentration for co-dilution of low molecular weight loading AN014 was 1000 nM, and 150 μg / mL of hIgG1 was used as a negative control. [Figure 4] This shows the viability of MDA-MB-231-Luc cells detected after co-culture treatment with MDA-MB-231-Luc / PBMC for 48 hours in 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC1-39, and HuSC1-39-L1-AN014. The starting concentration for co-dilution of low molecular weight loading AN014 was 1000 nM, and 150 μg / mL of hIgG1 was used as a negative control. [Figure 5] This shows the viability of HPAF-II-Luc cells detected after co-culture treatment of HPAF-II-Luc / PBMCs for 72 hours in 4A02-FCWT, 4A02-FCWT-L1-AN014, HuSC-39, and HuSC1-39-L1-AN014. The starting concentration for co-dilution of low molecular weight loading AN014 was 1000 nM, and 150 μg / mL of hIgG1 was used as a negative control. [Figure 6A] This study investigated the therapeutic effects of 4A02-FCWT-L1-AN014 in a nude mouse in vivo transplant tumor model of pancreatic cancer HPAF-II. Three days after cell inoculation, when the tumors had grown to approximately 100 mm³, tumor-bearing mice were randomly divided into groups and administered the treatments. The doses for 4A02-FCWT-L1-AN014 and HuSC1-39-L1-AN014 were 5 mg / kg, the nude antibody group received 4A02-FCWT and HuSC1-39 at 5 mg / kg, and the free low molecular weight AN014 dose was 2 mg / kg. These treatments were administered once a week for a total of two doses. [Figure 6B]This study investigated the therapeutic effects of HuSC1-39-L1-AN014 in a nude mouse in vivo transplant tumor model of pancreatic cancer HPAF-II. Three days after cell inoculation, when the tumors had grown to ~100 mm³, tumor-bearing mice were randomly divided into groups and administered the treatments. The doses for 4A02-FCWT-L1-AN014 and HuSC1-39-L1-AN014 were 5 mg / kg, the nude antibody group received 4A02-FCWT and HuSC1-39 at 5 mg / kg, and the free low molecular weight AN014 dose was 2 mg / kg. These treatments were administered once a week for a total of two weeks. [Figure 7] This shows the effects of serially diluted 4A02-FCWT-TM-4 and HuSC1-39-TM-4 in in vitro proliferation inhibition experiments of KRASG12D mutant HPAF-II cells, with inhibition curves and IC50 values ​​shown 3 days after drug treatment. [Figure 8] This study describes the therapeutic effect of TF-KRASG12D-I ADC HuSC1-39-TM-4 in an HPAF-II nude mouse transplant tumor model. Tumor-bearing mice were administered 10 mg / kg of nude antibody HuSC1-39 and HuSC1-39-TM-4 intravenously once a week for a total of three doses. [Figure 9] This describes the in vitro antitumor activity of the humanized antibody 4A02-HM8(FD40)-Topo1 inhibitor conjugate. The in vitro proliferative activity of FD40-LP1-D4, FD40-LP5-D4, and FD40-LP6-D4 against TF-negative breast cancer MDA-453, TF-high-expressing pancreatic cancer HPAF-II, BxPC3, lung cancer NCI-H1373, triple-negative breast cancer MDA-231, and HCC1806 cells is shown in the dose-response curve and IC50 value summary table. [Figure 10] This study describes the therapeutic effects of FD40-GGFG-Dxd, FD40-LP1-D4, FD40-LP5-D4, and FD40-LP6-D4 in a nude mouse in vivo transplant tumor model of lung cancer NCI-H1373. Eight days after cell inoculation, when the tumor had grown to ~200 mm3, tumor-bearing mice were randomly divided into groups and administered the treatment. 10 mg / kg of TF-NDC was administered intravenously once a week for a total of two times. [Figure 11]This study investigated the therapeutic effects of FD40-LP1-D4, FD40-LP5-D4, and FD40-LP6-D4 in a nude mouse in vivo transplant tumor model of pancreatic cancer HPAF-II. Seven days after cell inoculation, when the tumors had grown to approximately 150 mm³, tumor-bearing mice were randomly divided into groups and administered 10 mg / kg of TF-NDC intravenously, for a total of one dose. [Figure 12] This study describes the therapeutic effects of FD40-GGFG-Dxd (10 mg / kg) and FD40-LP5-D4 (10 mg / kg, 5 mg / kg, 2.5 mg / kg) in a nude mouse in vivo transplant tumor model of pancreatic cancer HPAF-II. Six days after cell inoculation, when the tumor had grown to ~200 mm3, tumor-bearing mice were randomly divided into groups and administered the drugs once in total. [Figure 13] This study describes the therapeutic effects of FD40-LP5-D4 (10 mg / kg, 5 mg / kg, 2.5 mg / kg) in a nude mouse in vivo transplant tumor model of triple-negative breast cancer HCC1806. Ten days after cell inoculation, when the tumor had grown to 200 mm³, tumor-bearing mice were randomly divided into groups and administered the drug once in total. [Figure 14A] Figure 14A shows the in vitro antitumor activity of HER2-NDC 1-G07-LP5, 1-G07-GGFG-Dxd, and T-Dxd. Figure 14A shows the antiproliferative activity of the drugs against gastric cancer NCI-N87 cells. [Figure 14B] Figure 14B shows the in vitro antitumor activity of HER2-NDC 1-G07-LP5, 1-G07-GGFG-Dxd, and T-Dxd. The antiproliferative activity of the drugs against breast cancer HCC1954 cells is shown. Dose-response curves and IC50 values ​​are also shown. [Figure 15] This study investigated the therapeutic effects of HER2-NDC 1-G07-LP5 and T-Dxd in an NCI-N87 nude mouse in vivo transplant tumor model. On day 16 after cell inoculation, when the tumor had grown to approximately 400 mm3, tumor-bearing mice were randomly divided into groups and administered the drug at a dose of 5 mg / kg, once a week for a total of two doses. [Figure 16]This study investigated the therapeutic effects of HER2-NDC 1-G07-LP5 and T-Dxd in an NCI-N87-Luc intracranial tumor model. Nine days after cell inoculation, tumor growth was monitored by in vivo imaging. The drugs were administered intravenously to divided groups at a dose of 5 mg / kg, once a week for a total of two doses. [Figure 17] This study investigated the therapeutic effect of TF-NDC FD40-LP5-D4 in an HCC1806-Luc intracranial tumor model. On day 7 after intracranial cell inoculation, tumor growth was monitored by in vivo imaging, and the drug was administered intravenously to separate groups at a dose of 5 mg / kg for a total of one dose. [Figure 18] These are the pharmacokinetic results of HER2-NDC 1-G07-LP5 and 1-G07-GGFG-Dxd administered intravenously to mice, with a dose of 1 mg / kg. [Figure 19] These are the pharmacokinetic results of TF-NDC FD40-LP5 and FD40-GGFG-Dxd administered intravenously to mice, with a dose of 1 mg / kg. [Figure 20] This shows the detection results for TF-NDC FD40-LP5 and TF-ADC HuSC1-39-MMAE in an in vitro blood-brain barrier permeability model. [Figure 21] This shows the drug analysis results of the FD40-LP5 scale-up conjugate in gram-scale batches. [Figure 22] This shows the results of body weight changes in an exploratory safety evaluation study experiment using cynomolgus monkeys with FD40-LP5. [Figure 23] This is the result of detecting blood coagulation indices in an exploratory safety evaluation study experiment using cynomolgus monkeys for FD40-LP5. [Figure 24] These are the results of detecting blood biochemical indicators in an exploratory safety evaluation study of FD40-LP5 using cynomolgus monkeys. [Figure 25] These are the results of hematological indicator detection in an exploratory safety evaluation study of FD40-LP5 using cynomolgus monkeys. [Figure 26]These are the pharmacokinetic (TK) detection results from an exploratory safety evaluation study of FD40-GGFG-Dxd and FD40-LP5 using cynomolgus monkeys. [Modes for carrying out the invention]

[0054] Through extensive and meticulous research, the inventors have constructed a novel linker for the first time. The antibody-drug conjugate constructed with the linker of the present invention exhibits remarkable tumor inhibitory effects against various cancer or tumor-derived cell lines, superior to positive controls. This indicates that the antibody-drug conjugate of the present invention can be used as a therapeutic agent for various solid tumors and hematological malignancies, and for the treatment of tumors or cancer. Based on this, the present invention was completed.

[0055] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs.

[0056] The term "approximately" can refer to a specific value or configuration within a set acceptable margin of error determined by those skilled in the art, which depends in part on how the value or configuration is measured or measured.

[0057] As used herein, the terms “contain” or “include” may be open, semi-closed, or closed. In other words, the terms also include “basically consist of” or “consist of.” Unless otherwise clearly indicated in the context, terms such as “contain,” “have,” and “include” in this entire specification and in the claims should be understood to have a comprehensive meaning, i.e., “includes, but not limited to,” rather than exclusive or exhaustive. Unless otherwise specified, “includes” includes “consist of.”

[0058] The term "alkyl group" refers to a saturated linear or branched aliphatic hydrocarbon group which consists of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C 1-20 The alkyl group has an alkyl group having 1 to 12 carbon atoms (i.e., C 1-12 It is an alkyl group, more preferably an alkyl group having 1 to 6 carbon atoms (i.e., C 1-6(Alkyl group). Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl Syl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, The group includes 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof. The alkyl group may be substituted or unsubstituted, and if substituted, it may be substituted by any available linking point, and the substituent is preferably selected from one or more of the following: D atom, halogen, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclic oxy group, hydroxyl group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclic group, aryl group, and heteroaryl group.

[0059] The term "alkylene group" refers to a divalent alkyl group, where an alkyl group is as defined above, and it consists of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C 1-20 The alkylene group has an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 An alkylene group), more preferably an alkylene group having 1 to 6 carbon atoms (i.e., C 1-6 The alkylene group is an alkylene group. Non-limiting examples include -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH2CH3)-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, etc. The alkylene group may be substituted or unsubstituted, and if substituted, it may be substituted by any available linking point, and the substituent is preferably selected from one or more of the following: D atom, halogen, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclic oxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclic group, aryl group, and heteroaryl group.

[0060] The term "alkoxy group" refers to -O-(alkyl), where alkyl is as defined above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy groups. The alkoxy group may be substituted or unsubstituted, and if substituted, it may be substituted by any available linking point, and the substituent is preferably selected from one or more of the following: D atom, halogen, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclic oxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclic group, aryl group, and heteroaryl group.

[0061] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic whole-carbocyclic (i.e., monocyclic cycloalkyl group) or polycyclic system (i.e., polycyclic cycloalkyl group) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 3 to 20-membered cycloalkyl group). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3 to 12-membered cycloalkyl group), and more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3 to 8-membered cycloalkyl group, e.g., C 3-7 A cycloalkyl group) is most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3 to 6 membered cycloalkyl group, for example C 3-6 It is a cycloalkyl group.

[0062] The term "aryl group" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aryl group) or a polycyclic aromatic ring system (i.e., a polycyclic aryl group) having a conjugated π-electron system, which has 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 6 to 14-membered aryl group). The aryl group is preferably an aryl group having 6 to 10 ring atoms (i.e., a 6 to 10-membered aryl group). The monocyclic aryl group is, for example, a phenyl group. Non-limiting examples of the polycyclic aryl group include naphthyl groups, anthracene groups, phenanthrene groups, etc. The aforementioned polycyclic aryl group further comprises a condensation group of a phenyl group with one or more heterocyclic groups or cycloalkyl groups, or a condensation group of a naphthyl group with one or more heterocyclic groups or cycloalkyl groups, where the linkage point is on the phenyl group or naphthyl group, and in this case, the number of ring atoms represents the number of ring atoms in the polycyclic aromatic ring system, and non-limiting examples include, [ka] This includes, among others.

[0063] The aryl group may be substituted or unsubstituted, and if substituted, it may be substituted by any available linking point, and the substituent is preferably selected from one or more of the following: D atom, halogen, alkyl group, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclic oxy group, hydroxy group, hydroxyalkyl group, oxo group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclic group, aryl group, and heteroaryl group.

[0064] The term "heteroaryl group" refers to a monocyclic heteroaryl ring (i.e., a monocyclic heteroaryl group) or a polycyclic heteroaryl ring system (i.e., a polycyclic heteroaryl group) having a conjugated π-electron system, wherein the ring contains at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized to form a nitrogen oxide, and the sulfur may optionally be oxidized to form a sulfoxide or sulfone, but does not contain -OO-, -OS-, or -SS-), and has 5 to 14 ring atoms (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) (i.e., a 5 to 14-membered heteroaryl group). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5 to 10-membered heteroaryl group), and more preferably a heteroaryl group having 5 or 6 ring atoms (i.e., a 5 or 6-membered heteroaryl group).

[0065] The term "cycloalkyloxy group" refers to a cycloalkyl-O- group, where cycloalkyl is as defined above. The term "heterocyclic oxy group" refers to a heterocyclic-O- group, where heterocyclic group is as defined above. The term "aryloxy group" refers to an aryl-O- group, where aryl group is as defined above. The term "heteroaryloxy group" refers to a heteroaryl-O- group, where heteroaryl group is as defined above. The term "alkylthio group" refers to an alkyl-S- group, where alkyl is as defined above. The term "halogenated alkyl group" refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above. The term "deuterated alkyl group" refers to an alkyl group substituted with one or more deuterium atoms, where alkyl is as defined above. The term "halogenated alkoxy group" refers to an alkoxy group substituted with one or more halogens, where alkoxy group is as defined above.

[0066] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxyl groups, where the alkyl group is as defined above. The term "halogen" refers to fluorine, chlorine, bromine, or iodine. The term "hydroxyl group" refers to -OH. The term "amino group" refers to -NH2. The term "cyano group" refers to -CN. N-ethyldiisopropylamine is abbreviated as DIEA. N,N-dimethylformamide is abbreviated as DMF. O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophospide is abbreviated as HATU. 1-Hydroxybenzotriazole is abbreviated as HOBt.

[0067] "Substituted" means that one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3, are independently substituted by a corresponding number of substituents. Those skilled in the art can determine whether substitution is possible or impossible without expending much effort (through experiment or theory). For example, an amino group or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (e.g., alkene) bond.

[0068] As used herein, the term “amino acid residue” refers to the group obtained by removing one H from the N-terminal -NH2 and the -OH from the C-terminal -COOH of an amino acid. Generally, the part of the chain containing the N-terminus and C-terminus of an amino acid (residue) is called the main chain, and the part that determines the specific type of amino acid is called the side chain. Generally, an amino acid residue is shown as -NH-CH(R)-CO-, where R is the side chain (amino acid side chain). Unless otherwise defined herein, amino acids include native and unnatural amino acids, including D-type and / or L-type amino acids. Examples of amino acids include, but are not limited to, Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), and Val(V). Preferably, in this specification, an amino acid is an amino acid selected from the group consisting of L-glycine (L-Gly), L-alanine (L-Ala), β-alanine (β-Ala), L-glutamic acid (L-Glu), L-aspartic acid (L-Asp), L-histidine (L-His), L-arginine (L-Arg), L-lysine (L-Lys), L-valine (L-Val), L-serine (L-Ser), and L-threonine (L-Thr). Furthermore, if the amino acid has two or more amino groups and / or two or more carboxyl groups, the term further includes groups formed by removing one H from -NH2 and one -OH from -COOH that are not on the same carbon atom, for example, the divalent group -C(O)-(CH2)2-C(COOH)-NH- formed by removing one H from -NH2 and one H from the non-α-positional -COOH of glutamic acid.

[0069] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is suitable as a drug when formed with an acid or base of the compound of the present invention. pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is one formed with an acid of the compound of the present invention. Acids suitable for salt formation include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0070] Compound of formula A [ka] In the formula, Q is a linker group for binding to the antibody. Z1 contains a glucose group containing Y hydroxyl groups, s is an integer between 0 and 10. n is an integer between 1 and 24. r is an integer between 0 and 10. X is a linking group, P1 is a polypeptide residue, P2 is a chemical bond or an AA-PAB structure, where AA is a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment in which 2 to 4 amino acids are linked by peptide bonds), and PAB is a p-aminobenzylcarbamoyl group. D is a drug.

[0071] Preparation method The following describes in more detail the methods for preparing compounds of the formula A structure of the present invention, but these specific methods do not limit the present invention. The compounds of the present invention can also be easily prepared by arbitrarily combining various synthesis methods described herein or known in the art, and such combinations can be easily performed by those skilled in the art.

[0072] Typically, in the preparation process, each reaction is carried out in a normally inert solvent at room temperature to reflux temperature (e.g., 0°C to 80°C, preferably 0°C to 50°C). The reaction time is usually 0.1 hours to 60 hours, preferably 0.5 to 48 hours.

[0073] The following general preparation routes can be used for the synthesis of compounds with the formula A structure of the present invention. [ka]

[0074] Furthermore, the preparation route for the antibody-drug conjugate is as follows: The interchain disulfide bonds of the antibody are reduced to generate 2n (e.g., 4) thiol groups. The substituted maleimide-based linker-drug conjugate of the present invention is crosslinked with the reduced antibody thiol groups to generate the corresponding antibody-drug conjugate.

[0075] antibody As used herein, the terms “antibody” or “immunoglobulin” refer to a heterotetrameric glycoprotein of approximately 150,000 daltons having the same structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes differs. Each heavy and light chain also has intrachain disulfide bonds arranged at regular intervals. Each heavy chain has a variable region (VH) at one end, followed by several constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the constant region of the light chain facing the first constant region of the heavy chain, and the variable region of the light chain facing the variable region of the heavy chain. Certain amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0076] As used herein, the term “variable” refers to a difference in the arrangement of specific portions of the variable region in an antibody, which forms the binding and specificity of different particular antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved portion of the variable region is called the framework region (FR). The natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folding configuration, connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folding structure. The CDRs of each chain are closely adjacent by FR regions and, together with the CDRs of another chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). While the constant region is directly involved in antibody binding to antigens, it also exhibits various effector functions, such as involvement in antibody-dependent cytotoxicity.

[0077] The "light chain" of vertebrate antibodies (immunoglobulins) can be assigned to one of two different classes (called κ and λ) according to the amino acid sequence of its constant region. The immunoglobulin can also be assigned to a different class according to the amino acid sequence of its heavy chain's constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

[0078] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located within the variable regions of the heavy and light chains, called variable regions (CDRs). These sections are divided into four framework regions (FRs), and the amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a cyclic structure, and the β-folds formed by the FRs between them are spatially close. The CDRs of the heavy chain and their corresponding CDRs of the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of similar antibodies, the amino acids that make up the FR or CDR region can be determined.

[0079] The present invention includes not only complete antibodies but also antibody fragments having immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention further includes the antibody fragments, derivatives, and analogs.

[0080] In the present invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies are useful antibodies such as chimeric and humanized monoclonal antibodies that contain human and non-human portions and can be obtained by standard DNA recombination techniques. Chimeric antibodies are molecules in which different portions originate from different animal species, for example, a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see U.S. Patents 4816567 and 4816397, which are incorporated herein by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5585089, which are incorporated herein by reference in their entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques known in the art.

[0081] In the present invention, the antibody may be monospecific, bispecific, triplicate, or have multiple specificities.

[0082] In the present invention, the antibody of the present invention further comprises its conserved variants, which means that, compared to the amino acid sequence of the antibody of the present invention, up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are substituted with similar or identical amino acids to form a polypeptide. These conserved variant polypeptides are preferably produced by substitution with amino acids according to Table A. [Table A]

[0083] Antibody preparation The DNA molecule sequences of the antibodies or fragments thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genome library screening. Furthermore, single-chain antibodies can be formed by fusing the coding sequences of the light and heavy chains.

[0084] Once the relevant sequence is obtained, it can be used to obtain large quantities of it using recombination. Typically, this is done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from host cells grown by conventional methods.

[0085] Furthermore, especially when the fragment length is relatively short, related sequences can also be synthesized by artificial synthesis methods. Typically, a very long fragment can be obtained by first synthesizing several small fragments and then concatenating them.

[0086] Currently, DNA sequences encoding the antibody (or its fragment or derivative thereof) of the present invention can be obtained entirely by chemical synthesis. These DNA sequences can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0087] The present invention further relates to vectors comprising the aforementioned appropriate DNA sequence and an appropriate promoter or regulatory sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0088] The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Preferred animal cells include, but are not limited to, CHO-S and HEK-293 cells.

[0089] Typically, the acquired host cells can be cultured and transformed under conditions suitable for the expression of the antibodies of the present invention. Next, the antibodies of the present invention are generated and obtained using conventional separation and purification methods well known to those skilled in the art, such as conventional immunoglobulin purification steps including protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0090] The obtained monoclonal antibodies can be identified by conventional methods. For example, the binding specificity of monoclonal antibodies can be measured using immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be measured, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0091] The antibodies of the present invention can be expressed intracellularly or at the cell membrane, or secreted extracellularly. If necessary, the recombinant proteins can be separated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional refolding, treatment with protein precipitants (salting-out methods), centrifugation, permeabilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), various other liquid chromatography techniques, and combinations thereof.

[0092] Antibody-drug conjugate (ADC) The present invention further provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention. Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, preferably by chemical coupling. Here, the effector molecule is preferably a drug having therapeutic activity. Furthermore, the effector molecule may be one or more of the following: a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0093] The antibody of the present invention can be coupled to the effector molecule via a coupling agent. Examples of the coupling agent include any one or more of the following: a non-selective coupling agent, a carboxyl group coupling agent, a peptide chain coupling agent, or a disulfide bond coupling agent. The non-selective coupling agent refers to a compound that covalently bonds the effector molecule and the antibody, such as glutaraldehyde. The carboxyl group coupling agent may be any one or more of the following: a cis-aconitic anhydride coupling agent (e.g., cis-aconitic anhydride), or an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0094] Specific residues on an antibody (e.g., Cys or Lys) are used to bind to various functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., glycol polymers), and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently bonded) to antibodies. Functional agents can be bound to antibodies directly or indirectly via linkers.

[0095] Typical coupling methods applicable to the present invention include K-Lock coupling methods and C-Lock coupling methods. In the K-Lock coupling method, the drug molecule is coupled to a lysine (K) residue in the antibody sequence, and in the C-Lock coupling method, the drug molecule is coupled to a cysteine ​​(C) residue in the antibody sequence.

[0096] Antibodies can form antibody-drug conjugates (ADCs) by being coupled to drugs. Typically, ADCs contain a linker located between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are usually susceptible to degradation in the intracellular environment, for example, at a site of interest that degrades the linker, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, such as peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, e.g., hydrazone linkers) and linkers that can be degraded under reducing conditions (e.g., disulfide linker). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by a protease.

[0097] Before binding to the antibody, the linker has an active reactive group that can react with specific amino acid residues, and binding is achieved via this active reactive group. Sulfhydryl-specific active reactive groups are preferred and include maleimide compounds, halogenated amides (e.g., iodine, bromide, or chlorine), haloesters (e.g., iodine, bromide, or chlorine), halomethyl ketones (e.g., iodine, bromide, or chlorine), benzyl halides (e.g., iodine, bromide, or chlorine), vinyl sulfones, pyridyl disulfide, mercury derivatives such as 3,6-bis-(mercurymethyl)dioxane whose counterion is acetate, chloride, or nitrate, and polymethylenedimethyl sulfide thiosulfonates. The linker may include, for example, maleimide bound to the antibody via thiosuccinimide.

[0098] The drug may be any cytotoxic, cell growth inhibitory, or immunosuppressive drug. In embodiments, the linker binds to the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug may have an amino group, carboxyl group, sulfhydryl group, hydroxyl group, or keto group capable of forming a bond with the linker. If the drug is directly bound to the linker, the drug has reactive active groups before binding to the antibody.

[0099] Useful drug classes include, for example, antitubulins, DNA ligation reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, and vinified alkaloids. Particularly useful examples of cytotoxic agents include, for example, DNA supraclution-binding reagents, DNA alkylation reagents, and tubulin inhibitors. Typical cytotoxic agents include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines) and vinca alkaloids.

[0100] In the present invention, a drug-linker can form an ADC in one simple step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step manner. For example, a cysteine ​​residue is reacted with the reactive moiety of the linker in the first step, and then in a subsequent step, the functional group on the linker is reacted with the drug to form an ADC.

[0101] Typically, functional groups on the linker are selected to facilitate specific reactions with appropriate reactive groups on the drug moiety. A non-limiting example is the use of an azide-based moiety to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently bonded to the linker via a 1,3-dipolar cyclic addition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides), isocyanates and isothiocyanates, and activated esters such as N-hydroxysuccinimidyl esters (suitable for reactions with amines and alcohols). These and other bonding strategies, as described in "Bioconjugation Techniques," second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art should understand that, for selective reactions of the drug moiety and linker, if a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used in both the linker and the drug.

[0102] The present invention provides a method for preparing an antibody conjugate (ADC), which may further include the step of conjugating an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0103] In certain embodiments, the method of the present invention includes the step of conjugating an antibody with a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further includes the step of conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to conjugate the drug moiety to the antibody via the linker.

[0104] In some embodiments, the antibody-drug conjugate (ADC) has the following molecular formula: [ka] Here, Ab is an antibody, LU is a linker, D is a drug, The subscript p is a value selected from 1 to 10, preferably from 1 to 8.

[0105] Drugs As used herein, “drug” broadly refers to a compound having a desired biological activity and a reactive functional group useful for the preparation of the conjugates described in this invention. The desired biological activity includes the diagnosis, cure, mitigation, treatment, and prevention of diseases in humans or other animals. Accordingly, as long as the necessary reactive functional group is present, the compound referred to in the term “drug” refers to drugs listed in the Official Chinese Pharmacopoeia, the American Homeopathic Pharmacopoeia, the Official Chinese Prescriptions Collection, or any supplements thereof. Typical drugs are listed in the Physician’s Reference Book of Drugs (PDR) and the U.S. Food and Drug Administration (FDA) Orange Book. As new drugs continue to be discovered and developed, it should be understood that these drugs also include “drugs” in the coupling drugs described in this invention.

[0106] The drugs that can be used to construct the ADC of the present invention include, but are not limited to, cytotoxic drugs (e.g., small molecule cytotoxic drugs).

[0107] The term "cytotoxic drug" refers to a substance that inhibits or blocks cell expression activity, cell function, and / or causes cell destruction. This term includes radioisotopes, chemotherapeutic agents, and toxins, such as low-molecular-weight toxins or enzymatically active toxins derived from bacteria, fungi, plants, or animals, and includes their fragments and / or variants. Examples of cytotoxic drugs include auristatin derivatives (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, meitansinoids, lysine, lysine A-chain, combretastatin, duocalmycin, dorastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, and vinbrassic acid. This includes, but is not limited to, tin, colchicine, dihydroxyanthracine dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modexin A chain, α-sarcin, geronin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents, and radioisotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioisotopes of Lu including Lu177. Antibodies can also couple prodrugs to their active form of anticancer prodrug activating enzymes.

[0108] Preferred low-molecular-weight drugs are compounds with high cytotoxicity, preferably monomethyl auristatin, gallicarin, meitansine derivatives, or combinations thereof, and more preferably selected from monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or combinations thereof.

[0109] Preferably, the drug refers to a cytotoxic drug used in cancer treatment, or a protein or polypeptide having a desired biological activity, such as toxins including absinthecin, lysine A, Pseudomonas exotoxin, and diphtheria toxin, other suitable proteins including tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, hemoglobin-derived growth factor, tissue plasminogen growth factor, and other biological response modifiers such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte maurophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, or other growth factors.

[0110] The preferred drugs of the present invention are maytansine or maytansinoids. Maytansine compounds inhibit cell proliferation by inhibiting tubulin microtubule formation. Maytansinoids are derivatives of maytansine. Both maytansine and maytansinoids have efficient cytotoxicity, but their clinical application in cancer treatment has significant limitations, mainly due to the low selectivity of this type of molecule for tumors. However, due to such high cytotoxicity, they are preferred drug moieties in antibody-drug conjugates. The structure of desacetylmytansine is shown below. [ka]

[0111] Another preferred drug of the present invention is auristatin peptide drugs. Auristatin peptide drugs are analogues of dolastatin 10, the latter being a biologically active polypeptide isolated from the body of the marine mollusk sea hare. Dolastatin 10 binds to tubulin (a binding domain similar to vincristine) and inhibits tubulin polymerization. Dolastatin 10, auristatin peptide PE, and auristatin peptide E are all linear polypeptides containing four amino acids (where three amino acids are specific to dolastatin compounds) and a C-terminal amide group. Both monomethyl auristatin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF), two representative auristatin peptide compounds, are preferred drugs for antibody-drug conjugates. [ka]

[0112] Another preferred drug of the present invention is pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs) or PBD dimers. PBDs are natural products produced by bacteria of the genus Streptomyces, and their unique property is the formation of non-deformable covalent adducts in DNA subgrooves, particularly in purine-guanine-purine sequences. The application of PBDs as part of small molecule strategies targeting DNA sequences, and as novel anticancer and antibacterial agents, is attracting increasing attention. By linking the C8 / C8' hydroxyl groups of two PBD units with a flexible carbon chain, the resulting dimers have enhanced biological activity. PBD dimers are thought to exert their biological activity by generating sequence-selective DNA damage, such as inverted 5'-Pu-GATC-Py-3' interchain crosslinks. These compounds have proven to be very potent cytotoxic drugs and can be used as candidate drugs for antibody-drug conjugates. [ka]

[0113] Another preferred drug of the present invention is the PNU-159682 derivative, which is the major active metabolite of nemorubicin in human liver microsomes, exhibiting 3000-fold increased activity compared to MMDX and doxorubicin. [ka]

[0114] In another embodiment, the drugs are not limited to the classes described above and further include all drugs that can be used in antibody-drug conjugates. In particular, these drugs that can coordinate to the amide bond of the linker, for example, cytotoxins that coordinate to a basic amine group (primary or secondary amine).

[0115] The term antibody-drug conjugate (ADC) refers to a monoclonal antibody or antibody fragment associated with a toxic drug having biological activity via a linking unit. The antibodies or antibody fragments described herein can be coupled to effector molecules in any way. For example, an antibody or antibody fragment can be conjugated to a toxic drug by chemical or recombinant methods. Chemical methods for preparing fusions or conjugates are known in the art. The method for coupling an antibody or antibody fragment to a drug must be able to link the antibody to the toxic drug without inhibiting the antibody or antibody fragment's ability to bind to the target molecule.

[0116] The present invention further provides a method for preparing an ADC, which may further include conjugating an antibody to a drug-linker compound (or a drug-linker compound such as LD-1 to LD-17 as shown in the present invention (linker-drug, LD)) under conditions sufficient to form an antibody-drug conjugate (ADC).

[0117] In certain embodiments, the method of the present invention includes conjugating an antibody to a linker compound under conditions sufficient to form an antibody-linker complex. In these embodiments, the method of the present invention further includes conjugating the antibody-linker complex to a drug moiety under conditions sufficient to covalently bond the drug moiety to the antibody via the linker.

[0118] The drug-to-antibody ratio (DAR), also known as the drug-to-antibody ratio, is the average number of drugs coupled to each antibody in the ADC. This can be, for example, within a range where each antibody couples to approximately 1 to 10 drugs, and in specific examples, within a range where each antibody couples to approximately 1 to 8 drugs, preferably within the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug-to-antibody ratio may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula of the ADCs disclosed herein includes a set of antibody-drug conjugates within the aforementioned ranges. In the embodiments disclosed herein, the drug-to-antibody ratio can be represented by n, which is a decimal or an integer. The drug-to-antibody ratio can be measured using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0119] In one embodiment of the present invention, a cytotoxic drug is coupled to an antibody via a coupling unit.

[0120] The amount of ligand-drug conjugate loading can be controlled by the following non-limiting methods: (1) Controlling the molar ratio of the drug linker fragment to the monoclonal antibody, (2) Controlling the reaction time and temperature, (3) Includes selecting different reaction reagents.

[0121] Pharmaceutical composition and administration method The antibody-drug conjugates provided by the present invention target specific cell populations and, by binding to cell surface-specific proteins (antigens), release the drug into the cells in an activated form via conjugate endocytosis or drug infiltration. Therefore, the antibody-drug conjugates of the present invention can be used to treat targeted diseases, and the antibody-drug conjugates mentioned above can be administered to subjects (e.g., humans) in therapeutically effective doses via appropriate routes. Subjects requiring treatment may be patients at risk of or suspected of having a disease related to the activity or expression level of a particular antigen. Such patients can be identified by conventional physical examinations.

[0122] The pharmaceutical composition may be administered to a subject using conventional methods known to those skilled in the medical field, depending on the type of disease requiring treatment or the site of the disease. The composition may also be administered by other conventional routes, such as orally, parenterally, inhalation spray, topically, rectally, nasally, orally, vaginally, or implantably. As used herein, the term “parenterally” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Furthermore, it may be administered via an injectable storage route, such as using injectable or biodegradable materials and methods with storage periods of one month, three months, or six months.

[0123] The injectable composition may contain various carriers such as vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). In the case of intravenous injection, water-soluble antibodies can be administered by drip infusion, thereby administering a drug preparation containing the antibody and physiologically acceptable excipients by infusion. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients. For example, intramuscular preparations such as sterile preparations in the appropriate soluble salt form of the antibody can be administered by dissolving pharmaceutical excipients such as aqueous injection solution, 0.9% saline, or 5% glucose solution.

[0124] When therapies are performed with the antibody-drug conjugate of the present invention, it can be delivered using conventional methods in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally using direct injection or infusion pumps. Other methods include various transport and carrier systems using conjugates and biodegradable polymers.

[0125] The pharmaceutical composition of the present invention comprises a safe and effective amount of the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Typically, drug formulations need to be matched to a method of administration, and the pharmaceutical composition of the present invention can be prepared in the form of a solution by preparing it using conventional methods, for example, physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably prepared under sterile conditions. The dose of the active ingredient is a therapeutically effective dose.

[0126] The effective dose of the antibody-drug conjugate described in the present invention can vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective dose can be determined by those skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the antibody-drug conjugate such as bioavailability, metabolism, and half-life, the severity of the disease in the patient being treated, the patient's body weight, the patient's immune status, and the route of administration. Typically, a good effect can be obtained when the antibody-drug conjugate of the present invention is administered daily at a dose of approximately 0.0001 mg to 50 mg / kg animal body weight (preferably 0.001 mg to 10 mg / kg animal body weight). For example, depending on the urgency of the treatment situation, the daily dose may be divided into several doses, or the dose may be reduced proportionally.

[0127] Dosage forms of the compounds of the present invention used for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable vector and any preservatives, buffers, or propellants as needed.

[0128] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0129] When the pharmaceutical composition is used, a safe and effective amount of the antibody-drug conjugate of the present invention is applied to a mammal (e.g., human) in need of treatment, where the dose at the time of administration is the effective dose to be considered, and for a person weighing 60 kg, the daily dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dose must also take into account factors such as the route of administration and the patient's health condition, and these are all within the scope of the skills of a skilled physician.

[0130] The main advantages of this invention are as follows: (1) SEC-HPLC detected that the monomer ratio of the complex was within the normal range (purity of all >90%), and the antibody-drug conjugate prepared with the novel linker of the present invention exhibited excellent solubility and drug potential, and no precipitation occurred during the coupling process.

[0131] (2) The linker of the antibody-drug conjugate of the present invention has good solubility, can significantly improve the water solubility of existing low molecular weight compounds, and improves the stability and uniformity of the conjugate after coupling with the antibody.

[0132] (3) It has a wide range of applications and can be used to obtain various antibody coupling drugs by coupling them with drugs of different mechanisms, which has the potential to improve the therapeutic range of existing coupling drugs.

[0133] (4) Compared to existing technologies, the hydrophilic side chain in the linker of the antibody-drug conjugate of the present invention has higher hydrophilicity, resulting in better coupling efficiency and applicability to coupling with different carrier drugs such as nanobodies.

[0134] (5) The complexes prepared by the linker of the present invention exhibit superior therapeutic effects in various in vitro and in vivo drug efficacy models and are superior to coupling drugs prepared by existing technologies.

[0135] (6) The complex prepared by the linker of the present invention has good safety in preclinical toxicity evaluations in cynomolgus monkeys.

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used solely for the purpose of illustrating the present invention and do not limit its scope. In the following examples, experimental methods that do not specify detailed conditions typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and quantities are calculated by weight.

[0137] Example 1. Synthesis of linker-STING agonist compound A1 (L1-AN014) Step 1: Preparation of Compound 3 [ka] Compound 1 (1.00 g, 1.96 mmol, 1.0 eq) and Compound 2 (720 mg, 2.95 mmol, 1.5 eq) are dissolved in 20 mL of anhydrous dichloromethane. HATU (900 mg, 2.37 mmol, 1.2 eq) and N,N-diisopropylethylamine (1.0 g, 7.83 mmol, 4.0 eq) are added, and the mixture is stirred at 25°C for 1 hour. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, and 40 mL of ethyl acetate and petroleum ether (1 / 2) are added to form a slurry. The residue is filtered, the filtrate is concentrated, and the concentrate is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile). The mixture is then lyophilized to obtain Compound 3 (1.3 g, 90%) as a colorless liquid. LC-MS (ESI) [M+H] + = 739.4.

[0138] Step 2: Preparation of Compound 4 [ka] Compound 3 (1.3 g, 1.80 mmol) is mixed with a hydrogen chloride-dioxane solution (6 M, 20 mL), then stirred at 25°C for 1 hour, and the reaction is detected by LC-MS. After the reaction is complete, the solvent is removed, the residue is diluted with dioxane (20 mL), concentrated three times, and dried to obtain the crude product Compound 4 (1.1 g, 100%) as a yellow liquid. LC-MS(ESI)[M+H] + = 627.3.

[0139] Step 3: Preparation of Compound 6 [ka] Compound 4 (900 mg, 1.44 mmol, 1.0 eq) is dissolved in 10 mL of anhydrous dioxane, and DIC (544 mg, 4.32 mmol, 3.0 eq) and HOSu (497 mg, 4.32 mmol, 3.0 eq) are added. The mixture is stirred at 25°C for 2 hours, then N,N-dimethylacetamide (5 mL), N,N-diisopropylethylamine (928 mg, 7.2 mmol, 5.0 eq), and Compound 5 (912 mg, 5.04 mmol, 3.5 eq) are added, and the mixture is stirred at 25°C for 1 hour. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, and the residue is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile). The mixture is then lyophilized to obtain Compound 6 (230 mg, 17%) as a white solid. LC-MS (ESI) [M+H] + =953.4.

[0140] Step 4: Preparation of Compound 9 [ka] Compound 7 (300 mg, 0.79 mmol, 1.0 eq) is dissolved in 5 mL of anhydrous dioxane, and DIC (200 mg, 1.58 mmol, 2.0 eq) and HOSu (183 mg, 1.58 mmol, 2.0 eq) are added. The mixture is stirred at 20°C for 2 hours, then saturated NaHCO3 solution (1 mL) and N,N-dimethylacetamide solution of compound 8 (173 mg, 1.19 mmol, 1.5 eq) (2 mL) are added. The mixture is then stirred at 20°C for 1 hour. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, and the residue is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile). The mixture is then lyophilized to obtain compound 9 (250 mg, 62%) as a white solid. LC-MS (ESI) [M+H-56] + = 449.2.

[0141] Step 5: Preparation of Compound 10 [ka] Compound 9 (200 mg, 0.40 mol, 1.0 eq) is dissolved in 5 mL of dichloromethane, and then trifluoroacetic acid (2 mL) is added. The reaction mixture is stirred at 20°C for 1 hour, and the reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, the residue is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), and lyophilized to obtain compound 10 (120 mg, 68%) as a white solid. LC-MS (ESI) [M+H] + = 449.2.

[0142] Step 6: Preparation of Compound 14 [ka] Compound 10 (76 mg, 0.17 mol, 1.2 eq) and compound AN014 (110 mg, 0.14 mol, 1.0 eq) were dissolved in 2 mL of N,N-dimethylacetamide, and then HATU (64 mg, 0.17 mol, 1.2 eq) and N,N-diisopropylethylamine (73 mg, 0.68 mmol, 4.0 eq) were added. The reaction mixture was stirred at 20°C for 1 hour, and the reaction was detected by LC-MS. After the reaction was complete, the reaction mixture was purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), lyophilized, and compound 11 (90 mg, 53%) was obtained as a white solid. LC-MS (ESI) [M+H] + = 1210.4.

[0143] Step 7: Preparation of compound A1 (L1-AN014) [ka] Compound 11 (80 mg, 0.066 mmol, 1.0 eq) and Compound 6 (94 mg, 0.099 mmol, 1.5 eq) were dissolved in a mixed solution of n-butanol / water (10 / 1.11 mL). Then, under nitrogen gas protection, sodium L-ascorbate (65 mg, 0.33 mmol, 5.0 eq) and copper sulfate pentahydrate (50 mg, 0.20 mmol, 3.0 eq) were added sequentially. The reaction mixture was stirred at 20°C under nitrogen gas protection for 2 hours, and the reaction was detected by LC-MS. After the reaction was complete, the mixture was concentrated, the residue was purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), and lyophilized to obtain compound L1-AN014 (12.11 mg, 8%) as a white solid. LC-MS (ESI) [1 / 2M+H] + = 1082.4.

[0144] Using PerkinElmer CHEMDRAW 22.2 software, the LogS parameters of compound 11 and compound A1 were calculated and compared. The LogS of compound 11 was -10.02, while that of compound A1 was -5.756, indicating an improvement of approximately 4-5 orders of magnitude in water solubility.

[0145] Example 2. Linker-KRAS G12DSynthesis of inhibitor compound A2(TM-4) Step 1: Preparation of Compound 3 [ka] Compound 1 (3.00 g, 9.55 mmol, 1.0 eq) and Compound 2 (1.02 g, 11.47 mmol, 1.2 eq) are dissolved in 50 mL of dioxane, and a solution of sodium bicarbonate (1.6 g, 19.1 mmol, 2.0 eq) in water (10 mL) is added. The mixture is stirred at 20°C for 20 hours. The reaction is detected by LC-MS. After the reaction is complete, the dioxane solvent is removed, the residue is diluted with ethyl acetate (50 mL), washed dropwise with water (50 mL), the pH of the aqueous phase is adjusted to acidity with aqueous citric acid, extracted with ethyl acetate (50 mL x 3), the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain the crude product Compound 3 (3.0 g) as a colorless liquid. LC-MS(ESI)[M+Na] + = 311.1.

[0146] Step 2: Preparation of Compound 5 [ka] Compound 3 (3.00 g, 10.41 mmol, 1.0 eq) and Compound 4 (1.28 g, 10.41 mmol, 1.0 eq) are dissolved in 70 mL of dichloromethane. HATU (4.75 g, 12.49 mmol, 1.2 eq) and N,N-diisopropylethylamine (5.38 g, 41.62 mmol, 4.0 eq) are added, and the mixture is stirred at 20°C for 1 hour. The reaction is detected by LC-MS. After the reaction is complete, the solvent is removed, and the mixture is purified by column chromatography (dichloromethane:methanol = 15:1) to obtain the crude product, Compound 5 (4.2 g, 100%), as a white solid. LC-MS (ESI) [M+Na] + = 416.3.

[0147] Step 3: Preparation of Compound 6 [ka] Compound 5 (4.2 g, 10.69 mmol, 1.0 eq) is dissolved in 60 mL of dichloromethane, trifluoroacetic acid (20 mL) is added, and the mixture is stirred at 20°C for 1 hour. The mixture is then concentrated under reduced pressure, the residue is dissolved in 50 mL of methanol, and saturated lithium hydroxide solution is added until the pH of the reaction mixture becomes basic. The mixture is then stirred at 20°C for 1 hour. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, purified by Prep-HPLC (0.1% aqueous ammonia / acetonitrile), and lyophilized to obtain compound 7 (2.2 g, 70%) as a yellow solid. LC-MS (ESI) [M+H] + =294.1.

[0148] Step 4: Preparation of Compound 16 [ka] Compound 17 (100 mg, 0.11 mmol, 1.0 eq) is dissolved in 5 mL of methanol, then Pd / C (10%, 20 mg) is added, and the mixture is stirred for 1 hour under a hydrogen gas environment at 20°C. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated to obtain compound 16 (90 mg, 93%) as a white solid.

[0149] Step 5: Preparation of Compound 9 [ka] Compound 7 (1.00 g, 3.25 mmol, 1.0 eq) and Compound 8 (0.66 g, 3.25 mmol, 1.0 eq) are dissolved in 20 mL of tetrahydrofuran, then N,N-diisopropylethylamine (1.26 g, 9.75 mmol, 3.0 eq) is added, and the mixture is stirred at 20°C for 16 hours. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), and lyophilized to obtain Compound 9 (1.2 g, 93%) as a white solid. LC-MS (ESI) [M+H-56] + =341.1.

[0150] Step 6: Preparation of Compound 10 [ka] Compound 9 (500 mg, 1.26 mmol, 1.0 eq) and Compound 6 (370 mg, 1.26 mmol, 1.0 eq) are dissolved in 5 mL of N,N-dimethylacetamide, and then HATU (575 mg, 1.51 mmol, 1.2 eq) and N,N-diisopropylethylamine (489 mg, 3.78 mmol, 3.0 eq) are added. The reaction mixture is stirred at 20°C for 1 hour, and the reaction is detected by LC-MS. After the reaction is complete, the reaction mixture is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), lyophilized, and Compound 10 (420 mg, 50%) is obtained as a white solid. LC-MS (ESI) [M+H-18] + = 654.3.

[0151] Step 7: Preparation of Compound 12 [ka] Compound 10 (150 mg, 0.22 mmol, 1.0 eq) and Compound 11 (136 mg, 0.44 mmol, 2.0 eq) are dissolved in 3 mL of N,N-dimethylacetamide, and then N,N-diisopropylethylamine (87 mg, 0.67 mmol, 5.0 eq) is added. The reaction mixture is stirred at 20°C for 5 hours. The reaction is detected by LC-MS. After the reaction is complete, the mixture is extracted with ethyl acetate, washed with water, washed with saturated brine, the organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and the residue is purified by silica gel column chromatography (dichloromethane / methanol = 15 / 1) to obtain Compound 12 (95 mg, 51%) as an orange-red solid. LC-MS (ESI) [M+H-56] + = 781.3.

[0152] Step 8: Preparation of Compound 14 [ka] Compound 12 (95 mg, 0.11 mmol, 1.0 eq) and Compound 13 (68 mg, 0.11 mmol, 1.0 eq) are dissolved in 2 mL of N,N-dimethylacetamide and 0.2 mL of pyridine. 1-hydroxybenzotriazole (15 mg, 0.11 mmol, 1.0 eq) and N,N-diisopropylethylamine (71 mg, 0.67 mmol, 5.0 eq) are added. The reaction mixture is stirred at 20°C for 16 hours. The reaction is detected by LC-MS. After the reaction is complete, the reaction mixture is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), lyophilized, and Compound 6 (90 mg, 61%) is obtained as a yellow solid. LC-MS(ESI)[M+H] + = 1298.5.

[0153] Step 9: Preparation of Compound 15 [ka] Compound 14 (90 mg, 0.07 mmol) is dissolved in 5 mL of dichloromethane, and trifluoroacetic acid (1 mL) is added. The reaction mixture is stirred at 20°C for 1 hour, and the reaction is detected by LC-MS. After the reaction is complete, the solvent is removed, and the residue is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile), and lyophilized to obtain compound 15 (45 mg, 52%) as a yellow solid. LC-MS (ESI) [M+H] + = 1243.4.

[0154] Step 10: Preparation of compound A2(TM-4) [ka] Compound 15 (45 mg, 0.036 mmol, 1.0 eq) is dissolved in 2 mL of anhydrous dioxane, and DIC (23 mg, 0.181 mmol, 5.0 eq) and HOSu (21 mg, 0.181 mmol, 5.0 eq) are added. The mixture is stirred at 20°C for 2 hours, then saturated NaHCO3 solution (0.2 mL) and a solution of compound 16 (173 mg, 1.19 mmol, 1.5 eq) in N,N-dimethylacetamide (1 mL) are added. The mixture is then stirred at 20°C for 2 hours. The reaction is detected by LC-MS. After the reaction is complete, the mixture is concentrated, and the residue is purified by Prep-HPLC (0.1% formic acid solution / acetonitrile). The mixture is then lyophilized to obtain compound TM-4 (11.02 mg, 14%) as a yellow solid. LC-MS (ESI) [1 / 2M+H] + = 1076.4.

[0155] Using PerkinElmer CHEMDRAW 22.2 software, the LogS parameters of compound 15 and compound A2 were calculated and compared. The LogS of compound 11 was -11.14, while that of compound A2 was -6.926, indicating an improvement of approximately four orders of magnitude in water solubility.

[0156] Example 3. Synthesis of linker-Topo1 inhibitor compound A4 (FD-LP1) Step 1: Synthesis of Compound 5 [ka] Compound 5a (200 mg, 0.596 mmol, 1.0 eq), HOSu (103.0 mg, 0.895 mmol, 1.5 eq), and DCC (246.1 mg, 1.193 mmol, 2.0 eq) were dissolved in tetrahydrofuran (5 mL). The reaction mixture was stirred at room temperature for 3 hours, and the completion of the reaction was monitored by LC-MS. The reaction mixture was then concentrated directly, and the residue was purified by silica gel column chromatography (DCM / MeOH = 93 / 7) to obtain compound 5 (160 mg, 37%) as a white solid. LCMS: [M + Na] + =455.1

[0157] Step 2: Synthesis of Compound 3 [Chemical formula] Compound 2 (247.5 mg, 0.466 mmol, 1.0 eq) and diisopropylethylamine (180.5 mg, 1.397 mmol, 3.0 eq) were dissolved in DMF (5 mL), stirred until clear, then Compound 1 (300 mg, 0.466 mmol, 1.0 eq) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (283.3 mg, 0.745 mmol, 1.6 eq) were added. After the addition was complete, the reaction mixture was stirred at 0 °C for 2 hours. After monitoring the completion of the reaction by LC-MS, the reaction mixture was directly pressurized and purified by reverse-phase purification (0.1% FA water / acetonitrile = 2 / 3) to obtain Compound 3 (420 mg, 81%) as a yellow solid. LCMS: [M+H] + = 1063.3

[0158] Step 3: Synthesis of Compound 4 [Chemical formula] Compound 3 (420 mg, 0.395 mmol, 1.0 eq) was dissolved in DMF (4 mL), cooled to 0 °C, then diethylamine (0.4 mL) was added. After the addition was complete, the reaction mixture was stirred at 0 °C for 30 minutes. After monitoring the completion of the reaction by LC-MS, the organic phase was concentrated to remove diethylamine. Acetic acid (420 mg) was dissolved in a small amount of DMF, added to the reaction system, shaken uniformly, the reaction system was directly pressurized, and purified by reverse-phase purification (0.1% FA water / acetonitrile = 7 / 3) to obtain Compound 4 (290 mg, 83%) as a yellow solid. LCMS: [M+H] + = 841.3

[0159] Step 4: Synthesis of Compound 6 [Chemical formula] Compound 4 (170 mg, 0.202 mmol, 1.0 eq), compound 5 (104.9 mg, 0.243 mmol, 1.2 eq) and diisopropylethylamine (52.3 mg, 0.404 mmol, 2.0 eq) were dissolved in DMF (5 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. After monitoring the completion of the reaction by LC-MS, the reaction mixture was directly pressurized and purified by reverse-phase purification (0.1% HCOOH / water / acetonitrile = 1 / 1) to obtain compound 6 (190 mg, 80%) as a white solid. LCMS: [M+H] + = 1158.4

[0160] Step 5: Synthesis of compound 7

Chemical formula

[0161] Step 6: Synthesis of compound 9

Chemical formula

[0162] Step 7: Synthesis of compound FD-LP1 [ka] Compound 9 (85 mg, 0.0753 mmol, 1.0 eq) and Compound 10 (107 mg, 0.1129 mmol, 1.5 eq) are dissolved in t-butanol:water = 1:1 (10 mL). Sodium L-ascorbate (74 mg, 0.3764 mmol, 5.0 eq) and copper sulfate pentahydrate (56 mg, 0.2258 mmol, 3.0 eq) are added. After the addition is complete, the reaction mixture is stirred under a nitrogen gas atmosphere at room temperature for 1 hour. After monitoring the completion of the reaction by LC-MS, the mixture is purified directly by Prep-HPLC (0.1% FA water / acetonitrile = 7 / 3) to obtain compound FD-LP1 (100 mg, 64%) as a pale yellow solid. LC-MS: [M / 2+H] + =1041.5 (half peak)

[0163] Example 4. Synthesis of linker-Topo1 inhibitor compound A9 (FD-LP5) Step 1: Synthesis of Compound 3 [ka] At 25°C, compound 1 (1 g, 2.70 mmol) is dissolved in anhydrous dichloromethane (50 mL), compound 2 (1.46 g, 8.10 mmol) is added, followed by PPTS (0.34 g, 1.35 mmol). The mixture is stirred at 45°C for 16 hours, and the mixture is concentrated under reduced pressure to obtain the crude product. The crude product is purified by forward silica gel column chromatography (ethyl acetate:petroleum ether = 0%~100%) to obtain compound 3 (1 g, 66.67%) as a white solid. MS m / z (ESI): 511.1 (M+Na) + .

[0164] Step 2: Synthesis of Compound 4 [ka] At 25°C, compound 3 (1 g, 2.04 mmol) is dissolved in ethanol (20 mL) and ethyl acetate (10 mL). Palladium carbon (1.18 g, 10% content) is added, the mixture is purged three times with hydrogen gas, and the reaction is carried out at room temperature for 2 hours under a hydrogen gas atmosphere. The reaction mixture is filtered and concentrated. The concentrate is purified by Prep-HPLC (water / acetonitrile) and lyophilized to obtain compound 4 (0.55 g, 62.16%) as a white solid. MS m / z (ESI): 421.1 (M+23) + .

[0165] Step 3: Synthesis of Compound 6 [ka] At 25°C, compound 4 (550 mg, 1.38 mmol) is dissolved in DMF (10 mL) solution, and compound 5 (462 mg, 1.79 mmol), HATU (605 mg, 2.07 mmol), and N,N-diisopropylethylamine (411 mg, 4.14 mmol) are added under ice bath. The resulting reaction mixture is stirred under ice bath for 1 hour. The organic phase is concentrated under reduced pressure to obtain the crude product, which is purified by forward silica gel column chromatography (ethyl acetate:petroleum ether = 0%~100%) to obtain the product compound 6 (277 mg, 28.83%, white solid). MS m / z (ESI): 816.2 (M+1) + .

[0166] Step 4: Synthesis of Compound 7 [ka] At 25°C, compound 6 (277 mg, 0.34 mmol) is dissolved in DMF (20 mL), and diethylamine (2 mL) of the reaction mixture is added at room temperature. The reaction mixture is stirred at room temperature for 0.5 hours. The mixture is concentrated under reduced pressure, purified by Prep-HPL (water / acetonitrile), and freeze-dried to obtain compound 7 (138 mg, 62.03%) as a white solid. MS m / z (ESI): 594.2 (M+1) + .

[0167] Step 5: Synthesis of Compound 9 [Chemical formula] At 25 °C, compound 7 (120 mg, 0.15 mmol) was dissolved in a DMF (10 mL) solution, and compound 8 (116 mg, 0.15 mmol), HATU (92 mg, 0.18 mmol), and N,N - diisopropylethylamine (80 mg, 0.45 mmol) were added under an ice bath. The resulting reaction mixture was stirred for 1 hour under an ice bath. The reaction solution was purified by Prep - HPL (1% FA water / acetonitrile) and freeze - dried to obtain compound 9 (80 mg, 31.09%) as a white solid. MS m / z (ESI): 1143.2 (M + 1) + .

[0168] Step 6: Synthesis of compound FD - LP5 [Chemical formula] Compound 9 (80 mg, 0.066 mmol) and compound 10 (100 mg, 0.10 mmol) were dissolved in a mixed solution of t - butanol / water (10 / 1, 11 mL), and then sodium L - ascorbate (69 mg, 0.34 mmol) and copper(II) sulfate pentahydrate (52 mg, 0.21 mmol) were sequentially added under nitrogen gas protection. The reaction solution was stirred at 20 °C for 0.5 hour under nitrogen gas protection, and the reaction was detected by LCMS. After completion of the reaction, the reaction solution was purified by Prep - HPLC (0.1% formic acid water / acetonitrile) and freeze - dried to obtain compound FD - LP5 (10 mg, 7%) as a white solid. LCMS (ESI) [1 / 2M + H] + = 1048.1.

[0169] Example 5. Synthesis of linker - Topo1 inhibitor compound A10 (FD - LP6) Step 1: Synthesis of compound 2 [Chemical formula] Under ice bath conditions, compound 1 (10.0 g, 0.0497 mol, 1.0 eq) is dissolved in acetic acid (60 mL), concentrated nitric acid (20 mL, con.) is slowly added, and the reaction mixture is stirred at 0°C for 1 hour. After monitoring the completion of the reaction by LC-MS, the reaction mixture is poured into ice water, a yellow solid precipitates, and after filtration, the filter cake is dried to obtain compound 2 (12.0 g, purity 90%, 88%) as a yellow solid. LC-MS: [M+H] + =245.9

[0170] Step 2: Synthesis of Compound 4 [ka] Compound 2 (5.0 g, 0.0203 mol, 1.0 eq) and Compound 3 (1.7 g, 0.0244 mol, 1.2 eq) are dissolved in DMF (60 mL). CuI (0.8 g, 0.0041 mol, 0.2 eq), bistriphenylphosphine palladium dichloride (2.1 g, 0.0030 mol, 0.15 eq), and triethylamine (10.3 g, 0.1015 mol, 5.0 eq) are added, and the mixture is reacted at 50°C for 4 hours. After monitoring the completion of the reaction by LC-MS, the organic phase is collected by extraction with ethyl acetate and dried over anhydrous sodium sulfate. The mixture is concentrated and separated and purified by column chromatography (PE:EA = 1:1) to obtain Compound 4 (4 g, purity 85%, 71%) as a brown solid. LCMS: [M + H] + = 236.1.

[0171] Step 3: Synthesis of Compound 5 [ka] Under ice bath conditions, dissolve compound 4 (4.0 g, 0.0170 mol, 1.0 eq) in EtOH:H2O=7:1 (80 mL), add sodium sulfide hydroxide (1.2 g, 0.0051 mol, 0.3 eq) and tin powder (4.0 g, 0.0340 mol, 2.0 eq), and slowly add concentrated hydrochloric acid (7 mL, 0.0850 mol, 5 eq) dropwise. React the mixture at 55°C for 1 hour. After monitoring the completion of the reaction by LC-MS, the reaction mixture was filtered by suction using diatomaceous earth while hot, eluted with a small amount of ethanol, the filtrates were combined, the filtrate was concentrated to dryness, dissolved in ethyl acetate, slowly diluted in aqueous sodium carbonate solution, and finally adjusted to pH=8. The mixture was filtered, the filtrate was collected, liquid-liquid separated, the organic phase was concentrated, and separated and purified by column chromatography (DCM:MeOH=20:1) to obtain compound 5 (1.6g, purity 70%, 29%) as a brown solid. LCMS:[M+H] + = 224.2.

[0172] Step 4: Synthesis of Compound 7 [ka] Compound 5 (1.1 g, 0.0049 mol, 1.0 eq) and Compound 6 (0.9 g, 0.0034 mol, 0.7 eq) are dissolved in NMP (10 mL), and p-toluenesulfonic acid monohydrate (0.93 g, 0.0049 mol, 1 eq) is added. After the addition is complete, the reaction mixture is stirred at 110 °C for 2 hours. After monitoring the completion of the reaction by LC-MS, the mixture is directly concentrated and separated and purified by column chromatography (DCM:MeOH=1:20) to obtain Compound 7 (0.6 g, purity 90%, 24%) as a brown oily solid. LCMS:[M+H] + = 451.1.

[0173] Stage 5: Compound 9 synthesis [ka] Under ice bath conditions, compound 7 (600.0 mg, 1.332 mmol, 1.0 eq) and compound 8 (2.5 g, 6.660 mmol, 5 eq) are dissolved in DMF (10 mL). Boron trifluoride ether solution (378.1 mg, 2.664 mmol, 2 eq) is added, and after addition, the reaction mixture is stirred at 30°C for 1 hour. The completion of the reaction is monitored by LC-MS. A small amount of ice water is added to quench the mixture, and it is directly concentrated. The crude product is separated and purified by column chromatography (DCM:MeOH=1:20) to obtain the crude product. This is then purified by reverse-phase column chromatography (0.1% FA water / acetonitrile=1 / 1) to obtain compound 9 (400 mg, 90% purity, 35%) as a brown solid. LCMS:[M+H] + = 759.2.

[0174] Step 6: Synthesis of Compound 11 [ka] Compound 9 (400.0 mg, 0.5272 mmol, 1.0 eq) is dissolved in DMF (5 mL), and diethylamine (462.7 mg, 6.3264 mmol, 12.0 eq) is added at room temperature. The reaction mixture is stirred for 0.5 hours. After monitoring the completion of the reaction by LC-MS, excess diethylamine is removed from the mixture under a reversed-phase column (0.1% FA water / acetonitrile = 9 / 1), and the mixture is purified with (water / acetonitrile = 7 / 3) to obtain compound 11 (307 mg, purity 90%, 97%) as a brown solid. LCMS: [M+H] + = 537.3.

[0175] Step 7: Synthesis of Compound 13 [ka] Compound 11 (240.0 mg, 0.4473 mmol, 1.0 eq) and Compound 12 (330.0 g, 0.5815 mmol, 1.3 eq) are dissolved in DMF (30 mL). N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)hexafluorophosphate urea (204.1 mg, 0.5368 mmol, 1.2 eq) and N,N-diisopropylethylamine (173.4 mg, 1.3419 mmol, 3 eq) are added. After the addition is complete, the reaction mixture is stirred at room temperature for 0.5 hours. After monitoring the completion of the reaction by LC-MS, the reaction mixture is purified by Prep-HPLC (0.1% FA water / acetonitrile = 7 / 3) to obtain Compound 13 (60 mg, 11%) as a yellow solid. LC-MS: [M+H] + =1086.3

[0176] Step 8: Compound FD-LP6 synthesis [ka] Compound 13 (60.0 mg, 0.0552 mmol, 1.0 eq) and Compound 10 (78.9 mg, 0.0828 mmol, 1.5 eq) are dissolved in t-butanol:water = 1:1 (10 mL). Sodium L-ascorbate (54.7 mg, 0.2760 mmol, 5.0 eq) and copper sulfate pentahydrate (41.4 mg, 0.1656 mmol, 3.0 eq) are added, and the reaction mixture is stirred under a nitrogen gas atmosphere at room temperature for 1 hour. After monitoring the completion of the reaction by LC-MS, the mixture is purified directly by Prep-HPLC (0.1% FA water / acetonitrile = 7 / 3) to obtain compound FD-LP6 (13 mg, 8%) as a yellow solid. LCMS:[M / 2+H] + = 1020.2 (half peak).

[0177] Example 6. Preparation of TF antibody, TF nanobody, and L1-AN014, TM-4 complex The heavy chain of the monoclonal antibody HuSC1-39 (derived from WO2018 / 036117 Al) targeting TF is as shown in SEQ ID NO. 10, and the light chain is as shown in SEQ ID NO. 11.

[0178] The nanobody-FC fusion protein that targets TF is 4A02-FCWT (where the CDR sequences are shown in SEQ ID NO.1-3, the VHH sequence is shown in SEQ ID NO.4, and the 4A02-FCWT fusion protein sequence is shown in SEQ ID NO.5).

[0179] Using a G25 desalting column, the antibody stock solution was replaced with 50 mM PB / 1.0 mM EDTA buffer (pH 7.0), 8 equivalents of TECP were added, and the mixture was stirred at 37°C for 2 hours to completely open the disulfide bonds between the antibody chains. Then, the pH of the reduced antibody solution was adjusted to 6.0 using phosphoric acid, and the water bath temperature was cooled to 25°C to prepare for the coupling reaction. The linker-drug conjugates prepared by the methods of Example 1 and Example 2 were dissolved in DMA, 12 equivalents of the linker-drug conjugate were aspirated and added dropwise to the reduced antibody solution, and DMA was added to bring the final concentration to 10% (V / V). The mixture was stirred at 25°C for 0.5 hours, and after the reaction was complete, the sample was filtered using a 0.22 μm membrane. Excess coupling low molecular weight molecules were purified and removed using a cross-flow ultrafiltration system. The buffer solution was 50 mM PB / 1.0 mM EDTA solution (pH=6.0). After purification, 6% sucrose was added, and the solution was stored in a refrigerator at -20°C. The absorbance values ​​were measured at 280 nM and 370 nM, respectively, using the UV method, and the DAR values ​​were calculated.

[0180] In this technical solution, precipitation does not occur during the coupling process of most linker-drug conjugates, and the DAR values ​​of the HuSC1-39 complex ADCs are all between 7 and 8, and the DAR values ​​of the 4A02-FCWT complex NDCs are all between 3.9 and 4. The DAR values ​​were measured using HIC-HPLC, RP-HPLC, or LCMS, and the polymer ratio of the conjugates detected by SEC-HPLC was within the normal range (purity was all >90%), indicating that the antibody-drug conjugates of the present invention have good solubility and drug potential and do not produce precipitation during the coupling process. Table 1 summarizes the preparation results of the four TF-ADCs and TF-NDCs. [Table 1]

[0181] Example 7. Preparation of TF humanized nanobody and HER2 nanobody-linker-Topo1 inhibitor FD-LP1, FD-LP5, and FD-LP6 complexes. Using the TF-targeting humanized nanobody-FC fusion protein 4A02-HM8-FCWT (code name FD40, sequence as shown in SEQ ID NO. 6), it is coupled with FD-LP1, FD-LP5, and FD-LP6 as described in Example 6, and the complex is purified and the detected DAR value is analyzed.

[0182] This technical solution prevents precipitation during the coupling process of most linker-drug conjugates, and the DAR values ​​of the 4A02-HM8-FCWT(FD40) complex NDCs are all between 3.6 and 3.9. The DAR values ​​were measured using HIC-HPLC, RP-HPLC, or LCMS, and the polymer ratio of the conjugates detected by SEC-HPLC was within the normal range (purity was all >90%), indicating that the antibody-drug conjugates of the present invention have good solubility and drug potential and do not produce precipitation during the coupling process. Table 2 summarizes the preparation results of the three TF-NDCs. [Table 2]

[0183] Similarly, using the HER2-targeting nanobody-FC fusion protein 1-G07-FCWT (sequence as shown in SEQ ID NO. 7), or the humanized HER2 nanobody-FC fusion proteins 1-G07-HM1-FCWT and 1-G07-HM3-FCWT (sequences as shown in SEQ ID NO. 8 and SEQ ID NO. 9), the FD-LP5 coupling reaction is performed as described in Example 6, the complex is purified, and the detected DAR value is analyzed. Table 3 shows the preparation results of the HER2-Topo1 inhibitor NDC. [Table 3]

[0184] Example 8. Activity detection of TF antibody / nanobody-L1-AN014 complex (TF-STING ADC, NDC) The cell lines used in the examples include triple-negative breast cancer cell line HCC1806, MDA-MB-231, pancreatic cancer cell line BxPC-3, and HPAF-II, which were purchased from the American Cell Culture and Cell Lineage Preservation Center (ATCC) and the Chinese Academy of Sciences Cell Bank, respectively, and cultured according to the corresponding instructions. Human peripheral blood mononuclear cell (PBMC) cryovials were provided by Jiangsu Xidil Biotechnology Co., Ltd.

[0185] 1 x 10 4 Individual tumor cells (using PBMC complete medium) are inoculated into 96-well plates and cultured overnight. PBMCs are then resuscitated individually in complete medium (RPMI1640 + 10% inactivated FBS + 1% penicillin / streptomycin + 1% sodium pyruvate + 1% glutamac). The following day, 1 × 10⁶ 5PBMCs (PBMC:tumor cells = 10:1) are added to a 96-well plate and incubated. Simultaneously, the test drug is diluted five-fold in the complete medium used to culture the PBMCs to obtain different concentrations. Here, HuSC1-39, 4A02-FCWT, HuSC1-39-L1-AN014, and 4A02-FCWT-L1-AN014 are all started at a concentration of 150 μg / mL, while AN014 and diABZI are started at a concentration of 1000 nM. The serially diluted test drugs are then added to the 96-well plate. After incubation at 37°C for an appropriate time, the cell supernatant is collected, and the viability of the tumor cells is detected using a firefly luciferase reporter gene detection kit or MTS reaction solution (MTS powder is purchased from Promega, catalog number G1111; PMS powder is purchased from Sigma, catalog number P9625).

[0186] Furthermore, based on the fact that CXCL10 and Interferon-γ (IFN-γ) are typical markers of STING pathway activation, the expression levels of CXCL10 (catalog number EK168, MULTI SCIENCES) and Interferon-γ (catalog number EK180, MULTI SCIENCES) in the supernatant of the co-cultured cells described above will be detected using an ELISA kit, and the activating effect of TF-STING ADC / NDC on the STING pathway will be evaluated.

[0187] As shown in Figure 1, triple-negative breast cancer MDA-MB-231+ human PBMCs were co-cultured for 48 hours. The nude antibody HuSC1-39, 4A02-FCWT slightly enhanced cytokine secretion compared to hIgG1, while the TF-STING ADC / NDC HuSC1-39-L1-AN014, 4A02-FCWT-L1-AN014 strongly induced the secretion of chemokines CXCL10 (Figure 1A) and Interferon-γ (Figure 1B). 50 The values ​​range from 0.01 to 0.05 nM, and as can be seen from this, its activation of the STING pathway is significantly higher than that of 1000 nM diABZI.

[0188] As shown in Figure 2, pancreatic cancer BxPC-3 + human PBMCs were co-cultured for 48 hours, and TF-STING ADC / NDC similarly showed a strong antitumor effect. Here, Figure 2A shows the killing EC against BxPC-3 cells. 50 Figure 2B shows <0.0002nM and <0.0004nM, indicating that TF-STING ADC / NDC strongly induces interferon-γ secretion activity, and EC 50 This indicates that the values ​​are approximately 1.12 nM and 1.21 nM.

[0189] As shown in Figures 3-5, in a TF-highly expressing tumor cell + PBMC co-culture system, TF-STING ADC / NDC showed more potent and effective tumor-killing activity compared to nude antibodies, and here, EC killing of HCC1806 cells after 24 hours. 50 The EC2 concentration was 0.014-0.066 μg / mL (Figure 3), and it was the EC2 50 This is approximately 0.0015 μg / mL (Figure 4), and represents the 72-hour EC-killing effect on HPAF-II cells. 50 This is approximately 0.01 μg / mL (Figure 5).

[0190] In an in vivo experiment, 5 × 10⁻¹⁰ 6 HPAF-II cells were inoculated into the backs of 6-week-old female Balb / c nude mice (Balb / c nude mice are purchased from Shanghai Sippr-BK Laboratory Animal Co., Ltd.), and the tumors were 150-200 mm in size. 3 After the animals had grown to a certain stage, they were randomly divided into groups and administered the drug, with each group containing eight tumors. As shown in Figure 6, administration of 3 mg / kg of 4A02-FCWT-L1-AN014 (once a week, for a total of two doses) significantly inhibited tumor growth compared to the solvent group or the low molecular weight loading therapy group alone.

[0191] Example 9. TF antibody / nanobody-TM-4 complex (TF-KRAS G12D Activity detection of inhibitors (ADC / NDC) The HPAF-II cell line used in the examples is purchased from the Cell Bank of the Chinese Academy of Sciences. The cells in the logarithmic growth phase are inoculated into a 96-well cell culture plate at a density of 1000-2000 cells per well at 150 μL / well, and cultured at 37°C and 5% CO2 for approximately 5 hours. Then, different concentrations of TF-ADC / NDCs (15 μg / mL to 0.00019 μg / mL) are added, and 2-4 duplicate wells, as well as corresponding solvent control and blank control wells, are set up for each drug concentration. After culturing for several days (confirming a sufficient number of cell divisions based on the cell growth rate), the culture medium is discarded, and 100 μL / well of MTS reaction solution (purchased from Promega, catalog number G3581) is added. The mixture is allowed to react at 37°C until the desired color intensity is reached, and the cell viability (OD490nm) of each group is measured and the cell viability is calculated according to the following formula. Survival rate = (OD administration - OD blank) / (OD control - OD blank) × 100%. The above data was analyzed using GraphPad Prism 8 software, and the IC of the above TF-NDC in different cell lines was also calculated. 50 Calculate the value.

[0192] KRAS G12D TF-KRAS using mutant pancreatic cancer HPAF-II cell line G12D The in vitro effects of the inhibitors ADC / NDC will be investigated. Cells in the logarithmic growth phase were inoculated into 96-well cell culture plates, and serially diluted HuSC1-39-TM-4 and 4A02-FCWT-TM-4 (15 μg / mL to 0.00019 μg / mL, n=3) were added to each. After 6 days of incubation, the culture medium was discarded, and MTS reaction solution (purchased from Promega, Cat#G3581) was added. Cell viability (OD490nm) was measured for each group.

[0193] As shown in Figure 7, HuSC1-39-TM-4 (DAR=7.56) and 4A02-FCWT-TM-4 (DAR=3.98) dose-dependently inhibit HPAF-II cell proliferation and IC 50 The values ​​are 0.0795 μg / mL and 0.0594 μg / mL, respectively.

[0194] In in vivo experiments, 5 × 10 6 HPAF-II cells were inoculated into the dorsal region of 6-week-old Balb / c female nude mice, and on day 6, the tumors grew to 150-200 mm. 3 After the animals had grown to a certain stage, they were randomly divided into groups and administered the treatment, with each group containing eight tumors. As shown in Figure 8, administration of 10 mg / kg of HuSC1-39-TM-4 (on days 6, 13, and 16) significantly inhibited tumor growth compared to the solvent group or the nude antibody HuSC1-39 treatment group.

[0195] Example 10. In vitro antitumor activity of TF nanobody-Topo1 inhibitor conjugate (TF-Topo1 inhibitor NDC) Refer to the method for detecting in vitro antitumor activity in Example 9. MDA-453, HPAF-II, BxPC3, HCC1806, MDA-231, and NCI-H1373 cells in the logarithmic growth phase were inoculated into 96-well cell culture plates at a density of 1000 to 3000 cells per well, at a rate of 150 μL / well. After culturing at 37°C and 5% CO2 for approximately 5 hours, different concentrations of TF-NDC FD40-GGFG-Dxd, FD40-LP1, FD40-LP5, and FD40-LP6 (15 μg / mL to 0.00019 μg / mL) were added to each group. After 6 days, the culture medium was discarded, and MTS reaction solution (purchased from Promega, cat#G3581) was added. The cell viability (OD490nm) of each group was measured.

[0196] As shown in Figure 9, FD40-GGFG-Dxd (DAR=4.0), FD40-LP1 (DAR=3.9), FD40-LP5 (DAR=3.9), and FD40-LP6 (DAR=3.8) dose-dependently inhibited cell proliferation in TF-high-expression cell lines HPAF-II, BxPC3, HCC1806, MDA-231, and NCI-H1373, and IC 50 The values ​​ranged from 0.0004 μg / mL to 0.0176 g / mL, and the IC for the TF-negative cell line MDA-453 was positive. 50The values ​​range from 4.5 g / mL to 5.1 g / mL. As can be seen from this, in in vitro scaling experiments, all TF-Topo1 inhibitor NDCs prepared in this invention exhibit TF target-dependent tumor cell killing effects.

[0197] Example 11. In vivo efficacy of TF nanobody-Topo1 inhibitor conjugate (TF-Topo1 inhibitor NDC). In vivo model for lung cancer NCI-H1373: 5x10 6 NCI-H1373 cells were inoculated into the dorsal region of 6-week-old Balb / c female nude mice, and tumors grew to ~200 mm on day 8. 3 After growth, the animals were randomly divided into groups, each containing 10 tumors. The drugs were administered once a week for a total of two times (on days 8 and 15). As shown in Figure 10, compared to the hIgG1-MMAE control group, administration of 10 mg / kg of FD40-GGFG-Dxd (DAR=4.0), FD40-LP1 (DAR=3.9), FD40-LP5 (DAR=3.9), and FD40-LP6 (DAR=3.8) significantly inhibited tumor growth. On day 32 of the experiment (17 days after drug discontinuation), FD40-LP5 and FD40-LP6 maintained superior tumor inhibitory effects. FD40-LP1 (P<0.001) showed a certain improvement in therapeutic efficacy compared to FD40-GGFG-Dxd (P=0.06).

[0198] In vivo model of pancreatic cancer HPAF-II: 5 x 10 6 When HPAF-II cells were inoculated into the dorsal region of 6-week-old Balb / c female nude mice, the tumor size was approximately 150 mm on day 7. 3 After the tumors had grown, the animals were randomly divided into groups and administered a total of 10 tumors to each group, for a total of one dose. As shown in Figure 11, compared to the hIgG1-MMAE control group, administration of 10 mg / kg of FD40-LP1, FD40-LP5, and FD40-LP6 significantly inhibited tumor growth. FD40-LP5 and FD40-LP6 showed superior therapeutic effects compared to FD40-LP1.

[0199] Similarly, in vivo model of pancreatic cancer HPAF-II: tumor size ~200mm on day 6 3 After growth, the animals were randomly divided into groups and administered the drug, with each group receiving 10 tumors and a total of one dose. As shown in Figure 12, administration of FD40-LP5 (10 mg / kg, 5 mg / kg, 2.5 mg / kg) all showed superior antitumor therapeutic effects compared to FD40-GGFG-Dxd at 10 mg / kg.

[0200] Triple-negative breast cancer (TNBC) HCC1806 model: HCC1806 cells in the logarithmic growth phase are placed in serum-free medium at a rate of 3 × 10⁶ per 200 L. 6 The spores were inoculated at a density of 1 in the mammary gland pads of 6-week-old Balb / c female nude mice, and the tumors grew to 200 mm. 3 After growing to maturity, the animals were randomly divided into groups, each receiving 10 tumors and a total of one dose. As shown in Figure 13, all treatment groups administered FD40-LP5 at doses of 10 mg / kg, 5 mg / kg, and 2.5 mg / kg showed excellent or good tumor treatment efficacy.

[0201] Example 12. In vitro and in vivo efficacy of the HER2 nanobody-Topo1 inhibitor conjugate (1-G07-LP5), and transcriptome analysis of tumor tissue genes. Referring to the method for detecting in vitro antitumor activity in Example 9, NCI-N87 cells (2000 cells / well) and HCC1954 cells (1000 cells / well) in the logarithmic growth phase were inoculated into a 96-well cell culture plate at 150 μL / well. After culturing at 37°C and 5% CO2 for approximately 5 hours, serially diluted concentrations of DS-8201 / T-Dxd (trastuzumab monoclonal antibody-GGFG-Dxd, DAR=8), 1-G07-GGFG-Dxd (DAR=3.9), and 1-G07-LP5 (DAR=3.4) were added. After 6 days, the culture medium was discarded, and MTS reaction solution (purchased from Promega, cat#G3581) was added. The cell viability (OD490nm) of each group was measured. The results are shown in Figures 14A (NCI-N87) and 14B (HCC1954), and in the detection of NCI-N87 cells, 1-G07-LP5 and 1-G07-GGFG-Dxd showed positive IC50. 50 The values ​​were 1.064 μg / mL and ~10 μg / mL, respectively, and in the detection of HCC1954 cells, the IC50 levels of 1-G07-LP5 and 1-G07-GGFG-Dxd were observed. 50 The values ​​were 0.58 μg / mL and 1.39 μg / mL, respectively, indicating that the in vitro antitumor activity of 1-G07-LP5 is higher than that of 1-G07-GGFG-Dxd.

[0202] Refer to the in vivo efficacy detection method in Example 11, and 200 μL of 5 × 10⁶ 6 A PBS-matrix gel (PBS:standard concentration matrix gel = 1:1) suspension containing NCI-N87 cells is inoculated subcutaneously into the lateral dorsal region of female nude mice (Balb / c nude, 5-6 weeks old). The tumor volume is approximately 400 mm². 3When reaching (on the 16th day), randomly group according to the size of the tumor volume and the body weight of nude mice (n = 8), and use doses of 5 mg / kg of T-Dxd (DAR = 8) and 5 mg / kg of 1-G07-LP5 (DAR = 3.4) respectively, and administer a total of 2 times via the tail vein (on the 16th day and the 23rd day), and at the same time set hIgG1-vc-MMAE as the negative control. The results are as shown in Figure 15. Compared with the control group, 5 mg / kg of T-Dxd (DAR = 8) inhibited tumor growth, 5 mg / kg of 1-G07-LP5 (DAR = 3.4) brought about obvious tumor shrinkage, and the difference in drug efficacy between the two groups had statistical significance (p < 0.05).

[0203] Example 13. Therapeutic effects of HER2-NDC 1-G07-LP5 and TF-NDC FD40-LP5 in a nude mouse intracranial tumor model Construct the NCI-N87 intracranial model to detect 1-G07-LP5. Resuspend NCI-N87-luc cells in PBS and adjust the concentration to 1×10 8 / mL, and use a microsyringe to aspirate 5 μL of cells (5×10 5 cells) for inoculation. Select female Balb / c nude mice at 6 - 7 weeks old, after anesthetizing with avertin, fix them in a stereotactic apparatus. Centering on the frontal suture of the mouse head, move the microinjection needle 2 mm to the right and 0.6 mm upward, puncture the skull with a triangular needle, then advance the needle 4 mm downward and pull it up 1 mm, and inject the tumor cells at a uniform speed. Seal the puncture site of the skull with bone wax, and finally suture the scalp. 9 days later, inject D-luciferin (150 mg / kg) intraperitoneally, use a small animal fluorescence / CT in vivo imaging system to collect in vivo fluorescence images and count the fluorescence signal intensity in the brain. The fluorescence signal intensity Radiance (p / sec / cm 2The mice are grouped based on their body weight ( / sr) and other factors. A solvent control group, a 5 mg / kg 1-G07-LP5 group, and a 5 mg / kg T-Dxd group are established. The drug is administered intravenously in the tail vein, with a second dose given one week later, for a total of two doses. In vivo fluorescence images are collected every 1-2 weeks using a small animal fluorescence / CT in vivo imaging system, and the fluorescence signal intensity (Radiance (p / sec / cm)) of the brain is collected. 2 Tumor growth curves were created by statistically analyzing the body weight of nude mice and the sr(s)

[0204] Construct an intracranial model of HCC1806 to detect FD40-LP5. Referring to the above modeling and experimental methods, use a microsyringe to inject 5 L of HCC1806-luc cells (5 × 10⁶) into the cranial cavity at a uniform rate. 5 The mice were injected with 5 mg / kg of hIgG1-MMAE (DAR=3.9). After 7 days, they were randomly divided into groups based on fluorescence signal intensity and mouse body weight (n=4). The mice were then given 5 mg / kg of FD40-LP5 (DAR=3.9) via tail vein, for a total of one dose. In vivo fluorescence images were collected weekly using a small animal fluorescence / CT in vivo imaging system, and brain fluorescence signal intensity and nude mouse body weight were statistically recorded. The results are shown in Figure 17, and compared to the hIgG1-MMAE group, 5 mg / kg of FD40-LP5 completely inhibited tumor growth or caused partial regression.

[0205] Example 14. Detection of pharmacokinetics of HER2-NDC and TF-NDC in mouse in vivo. Serum sample collection: HER2-NDC, 1 mg / kg of 1-G07-GGFG-Dxd, and 1 mg / kg of 1-G07-LP5 were administered intraveinally into the tail vein of 8-week-old female Balb / c mice. Approximately 100 μL of blood was collected from the orbit at 0 minutes, 5 minutes, 30 minutes, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, and 192 hours after administration. The blood was allowed to stand at room temperature for 30 minutes, then at 4°C for 3-4 hours, and centrifuged at 1500 rpm for 15 minutes to collect the upper layer serum. Detection of antibody-drug conjugate concentration by ELISA: Dilute anti-Dxd antibody (Abmax Biotechnology, Cat#05-0191-L, equivalent LP5 affinity to Dxd) to 2.5 μg / mL using coating solution, coat ELISA plates with 100 μL / well, seal with a sealing membrane, and incubate overnight at 4°C. Remove unbound antigen, add 200 μL / well of 3% BSA blocking solution prepared in PBS, and block at room temperature for 2 hours. Remove blocking solution, add the serum sample to be tested (3-fold serial dilution), and simultaneously add 100 μL / well of a standard sample (starting at 333.33 ng / mL) (3-fold serial dilution), and incubate at room temperature for 2 hours. Remove unbound antibody, add 100 μL / well of HRP-labeled secondary antibody diluted according to 1:8000, and incubate at room temperature for 1 hour. Unbound secondary antibody is removed, and 150 μL / well of TMB chromogenic solution is added. The mixture is allowed to develop color at room temperature in the dark for approximately 5 minutes. 50 μL / well of ELISA stop solution is added to stop the color reaction. The OD value at a wavelength of 450 nM is measured, and the serum drug concentration is analyzed and calculated. The obtained serum drug concentrations at different time points are entered into Phoenix software, and the drug concentration-time related parameters are analyzed using a non-compartment model. The results are shown in Figure 18, and after intravenous administration of 1 mg / kg, the mean Cmax values ​​of 1-G07-GGFG and 1-G07-LP5 were 18.6 μg / mL and 17.5 μg / mL, respectively. 1 / 2 The average values ​​were 34.6 hours and 35.9 hours, respectively, and AUC 0-tThe average values are 380.5 hours×μg / mL and 456.3 hours×μg / mL respectively, and the MRT last The average values are 33.9 hours and 43.1 hours respectively. As can be seen from the results, LP5 has improved stability in the in vivo circulation system compared with GGFG-Dxd.

[0206] Similarly, the drugs of TF-NDC, 1 mg / kg of FD40-GGFG-Dxd, and 1 mg / kg of 1-FD40-LP5 are intravenously injected into the bodies of 8-week-old female Balb / c mice via the tail vein respectively. At 0 minute, 5 minutes, 30 minutes, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, and 192 hours after administration, about 100 μL of blood is collected from the eye socket and used for detection and analysis after treatment. The results are as shown in Figure 19. After intravenous administration of 1 mg / kg, the average values of Cmax of FD40-GGFG-Dxd and FD40-LP5 are 16.5 μg / mL and 16.75 μg / mL respectively, and the T 1 / 2 The average values are 45.56 hours and 52.29 hours respectively, and the AUC 0-t The average values are 272.88 hours×μg / mL and 531.75 hours×μg / mL respectively, and the MRT last The average values are 34.92 hours and 49.12 hours respectively. As can be seen from the results, LP5 has significantly improved stability in the in vivo circulation system compared with GGFG-Dxd.

[0207] Example 15. Permeability of TF-NDC FD40-LP5 and TF-ADC in an in vitro blood-brain barrier (BBB) model The cells used in this example were obtained from Wuhan Pricella Biotechnology Co., Ltd. and cultured according to the corresponding instructions, including C8-D1A and b.End3. Construction of the in vitro BBB model: A 6.5 mm diameter, 3 μm pore chamber (6.5 mm, Corning, Cat#3415) was coated with 100 μg / mL mouse tail type I collagen at 37°C for 1 hour. Mouse brain astrocytes C8-D1A were resuspended in DMEM / F12 complete medium. The 24-well plate and chamber were inverted, and 50 μL of C8-D1A cell suspension was dropped onto the bottom of the chamber, with a final cell density of 1 × 10⁶. 5 cells / cm 2 The bottom of the 24-well plate is covered with a lid and incubated in an incubator for 3 hours. The chamber and 24-well plate are returned to their original positions, the culture medium is replenished, and the cells are cultured for another 48 hours. The mouse microvascular endothelial cells b.End3 are resuspended in DMEM / F12 complete medium. 100 μL of b.End3 cell suspension is added to the upper layer of the chamber, and the final cell density is 2 × 10⁶. 5 cells / cm 2 The cells are placed in an incubator and incubated for 5 hours, then the culture medium is replenished and cultured for another 96 hours. Cellular confluence in the chamber is confirmed by staining with 0.2% crystal violet.

[0208] Permeability detection: Remove the culture medium and add 700 μL of fresh medium to the bottom of the chamber. Add 100 μg / mL of different drugs to the top of the chamber and incubate in an incubator. At time points 6 and 24, collect 120 μL of the medium from the bottom of the chamber and replenish with 120 μL of fresh medium as appropriate. For TF-NDC and TF-ADC drugs, detect antibody concentration using ELISA. The results are shown in Figure 20, and at two time points, 6 hours and 24 hours, the BBB transmittance of FD40-LP5 was approximately twice that of TF-ADC HuSC1-39-MMAE.

[0209] Example 16. Preparation and Stability Detection of FD40-LP5 Scale-Up Batch Complex Based on Example 7, FD40-LP5 was scaled up and prepared in batches by adjusting and optimizing the coupling reaction and complex purification conditions. The coupling reaction was performed using 860 mg of FD40 antibody, with a TECP / antibody molar ratio of 2.8, an LP5 / antibody molar ratio of 7.0, and 10% organic solvent DMA. The reaction conditions were reduction at 22°C for 18 hours followed by coupling at 22°C for 0.5 hours. The overall yield of the complex was 81%, DAR = 4.0, monomer ratio was 97.39, and residual low molecular weight was <0.06%. Figure 21 shows the SEC and LC-MS detection data of the batch-scaled FD40-LP5 complex.

[0210] The FD40-LP5 complex from this batch was used to repeatedly perform freeze-thaw stability and thermal stability tests. The results are shown in Table 4, indicating that the FD40-LP5 complex exhibits excellent freeze-thaw stability and thermal stability. [Table 4]

[0211] Example 17. Safety evaluation of FD40-LP5 exploration in cynomolgus monkeys. Two cynomolgus monkeys (one female and one male) were administered FD40-LP5 as a single intravenous dose of 10 mg / kg, followed by 21 consecutive days of observation. On the 22nd day, FD40-LP5 was administered again intravenously at a dose of 30 mg / kg, and observation continued for another 21 days (total of 42 days). The results showed no obvious drug-related changes in the animals' clinical condition at any of the doses, and no minor and reversible changes were observed in feeding or body weight indicators. As shown in Figure 22, during the study period, especially at high doses, the animals' body weight decreased slightly and reversibly (5-10%). As shown in Figure 23, no significant drug-related alterations were observed in the animals' blood coagulation indicators at each dosage level. As shown in Figure 24, no significant drug-related alterations were observed in the animals' blood biochemical indicators at each dosage level. As shown in Figure 25, no significant drug-related alterations were observed in the animals' hematological indicators at each dosage level.

[0212] Example 18. Pharmacokinetic (TK) detection of FD40-GGFG-Dxd and FD40-LP5 in cynomolgus monkeys Study K2477: Two cynomolgus monkeys (one female, one male) were used. A single intravenous dose of 10 mg / kg of FD40-GGFG-Dxd (numbered as the 10 mg / kg dose group) was administered, and on day 22, another intravenous dose of 30 mg / kg of FD40-GGFG-Dxd (numbered as the 30 mg / kg dose group) was administered. Before each administration, blood was collected from the forelimb or hindlimb vein at 5 minutes, 1 hour, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, 240 hours, 336 hours, and 504 hours after administration, and serum was prepared. Pharmacokinetic detection was performed referring to the method in Example 14.

[0213] Study K2504: Two cynomolgus monkeys (one female, one male) were used. A single intravenous dose of FD40-LP5 at 10 mg / kg (numbered as the 10 mg / kg dose group) was administered, and on day 22, another intravenous dose of FD40-LP5 at 30 mg / kg (numbered as the 30 mg / kg dose group) was administered. Before each administration, blood was collected from the forelimb or hindlimb vein at 5 minutes, 1 hour, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, 240 hours, 336 hours, and 504 hours after administration, and serum was prepared.

[0214] Pharmacokinetic detection was performed using the method of Example 14. The results are shown in Figure 26, and the AUC of FD40-GGFG-Dxd and FD40-LP5 was measured after intravenous administration of 10 mg / kg. 0-t The mean values ​​were 4725.19 hours × μg / mL and 7527.15 hours × μg / mL, respectively, after intravenous administration of 30 mg / kg, and the AUC of FD40-GGFG-Dxd and FD40-LP5. 0-tThe average values ​​were 14822.3 hours × μg / mL and 24615.3 hours × μg / mL, respectively. As can be seen from these results, LP5 exhibits significantly improved stability in the internal circulatory system of cynomolgus monkeys compared to GGFG-Dxd.

[0215] In summary, the study of the relevant examples clearly demonstrates the following: 1. TF-STING ADC / NDC prepared with the novel linker A of the present invention exhibits excellent antitumor effects in triple-negative breast cancer and pancreatic cancer models by activating the STING signaling pathway, promoting tumor-immune cell interactions, and inducing the secretion of CXCL10 and IFN.

[0216] 2. TF-KRAS prepared by the novel linker A of the present invention G12D -I ADC / NDC is KRAS G12D It exhibits favorable antitumor effects in a mutant pancreatic cancer model.

[0217] 3. TF-Topo1 inhibitor NDCs prepared with the novel linker A of the present invention are used in KRASG12C mutant lung cancer models and KRAS G12D It exhibits favorable antitumor effects in mutant pancreatic cancer models and triple-negative breast cancer models.

[0218] 4. The HER2-Topo1 inhibitor NDC prepared with the novel linker A of the present invention exhibits good antitumor effects in a gastric cancer model and is superior to trastuzumab-deruxtecan (T-Dxd).

[0219] 5. In intracranial tumor models, TF-NDC and HER2-NDC prepared with novel linker A exhibit good BBB permeability, good antitumor efficacy against intracranial tumors, and are superior to trastuzumab-deruxtecan (T-Dxd).

[0220] 6. In head-to-head comparative mouse pharmacokinetic experiments, HER2-NDC and TF-NDC prepared with the novel linker A exhibited higher in vivo half-lives and plasma exposures compared to the known linker (GGFG-Dxd). Similarly, in the detection of in vivo toxic metabolism (TK) in cynomolgus monkeys, the mean plasma exposure of FD40-LP5 was significantly higher than that of FD40-GGFG-Dxd.

[0221] 7. The novel linker A of the present invention has a wide range of applications and is suitable for antibodies, nanobodies, and loading compounds having different mechanisms of action that are coupled to different targets.

[0222] 8. The coupling purification technique for preparing NDC / ADC using the novel linker A of the present invention is stable and useful for industrial processes.

[0223] 9. FD40-LP5 prepared with the novel linker A of the present invention has extremely high drug discovery potential, exhibits excellent water solubility and homogeneity, and demonstrates repeated freeze-thaw stability and thermal stability.

[0224] 10. FD40-LP5 prepared with the novel linker A of the present invention demonstrates good safety in exploratory toxicity evaluation studies using cynomolgus monkeys.

[0225] The present invention includes TF nanobodies, HER2 nanobodies, and TF monoclonal antibody sequences. SEQ ID NO.1:4-A02 CDR1 ETISSTYI SEQ ID NO.2:4-A02 CDR2 ISGDGVTH SEQ ID NO.3:4-A02 CDR3 YAAGRWNH SEQ ID NO.4:4A02 VHH QVQLVESGGGLVQPGGSLSLSCTASETISSTYIMGWHRRSPGKERELVAVISGDGVTHYADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYCYAAGRWNHWGQGTQVTVSS SEQ ID NO.5:4A02-FCWT QVQLVESGGGLVQPGGSLSLSCTASETISSTYIMGWHRRSPGKERELVAVISGDGVTHYADFVKGRFVISRDNAKNAVYLKMNFLTPEDTAVYYCYAAGRWNHWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.6:4A02-HM8-FCWT QVQLVESGGGLVKPGGSLRLSCTASETISSTYIMGWHRQAPGKGRELVAVISGDGVTHYADFVKGRFTISRDNAKNTVYLQMNFLRAEDTAVYYCYAAGRWNHWGQGTMVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.7:1-G07-FCWT QVQLVESGGGKAQPGGSLRLSCVASRITFRHYDLSWYRQAPGQERELVATVTNGGVITYADSVKGRFTISRDNAKNTVQLQMNNLKPEDTAVYYCNAVWVYLQRLTQNKKENDYWGPGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.8:1-G07-HM1-FCWT QVQLVESGGGLVQPGGSLRLSCSASRITFRHYDLSWYRQAPGKGRELVATVTNGGVITYADSVKGRFTISRDNAKNTVYLQMNNLRAEDTAVYYCNAVWVYLQRLTQNKKENDYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.9:1-G07-HM3-FCWT QVQLVESGGGLVKPGGSLRLSCAASRITFRHYDLSWYRQAPGKGRELVATVTNGGVITYADSVKGRFTISRDNAKNTVYLQMNNLRAEDTAVYYCNAVWVYLQRLTQNKKENDYWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.10: HuSC1-39 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMNWVRQMPGKGLEWMGMIYPADSETRLNQKFKDQATLSVDKSISTAYLQWSSLKASDTAMYYCAREDYGSSDYWGQGTTVTVSS SEQ ID NO.11: HuSC1-39 VL DIQLTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKSPKIWIYGISNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKSSFPWTFGGGTKVEIK

[0226] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. A compound or its stereoisomer or a pharmaceutically acceptable salt thereof, The compound has a structure as shown in formula A, 【Transformation 56】 In the formula, Q is a linker group for binding to the antibody. Z1 contains a glucose group containing Y hydroxyl groups, s is an integer between 0 and 10. n is an integer between 1 and 24. r is an integer between 0 and 10. X is a linking group, P1 is a polypeptide residue, P2 is a chemical bond or an AA-PAB structure, where AA is a dipeptide, tripeptide, or tetrapeptide fragment (i.e., a fragment in which 2 to 4 amino acids are linked by peptide bonds), and PAB is a p-aminobenzylcarbamoyl group. D is a compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that D is a drug.

2. The linker base Q is selected from the following: 【Chemistry 57】 Here, A represents an optionally substituted C3-C8 alkylene group, C3-C8 alkenyl group, C3-C8 alkynyl group, C3-C6 cycloalkenyl group, C3-C8 cycloalkyl group, or an optionally substituted diethylene glycol to octaethylene glycol acyl group; Ar represents an optionally substituted C5-C6 aryl group or heteroaryl group; and "*" indicates that -C=O- forms an amide bond with an amino group. The compound described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof.

3. The P1 is ＮＨ -Val-Cit- Ｃ＝Ｏ , ＮＨ -Val-Ala- Ｃ＝Ｏ , ＮＨ -Ala-Ala-Ala- Ｃ＝Ｏ , ＮＨ -Ala-Ala- Ｃ＝Ｏ , ＮＨ -Gly-Gly-Phe-Gly- Ｃ＝Ｏ , ＮＨ -Val-Lys- Ｃ＝Ｏ characterized by being selected from the group consisting of The compound described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof.

4. The aforementioned X is, 【Chemistry 58】 Characterized by being selected from The compound described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof.

5. The structure of Z1 is, 【Chemistry 59】 Characterized by being selected from the group consisting of The compound described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof.

6. The aforementioned D is characterized by being a cytotoxic small molecule drug selected from the group consisting of STING agonists, KRAS-G12D inhibitors, tubulin inhibitors, topoisomerase inhibitors, and DNA binders. The compound described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof.

7. The structure of formula A is characterized by being selected from the group consisting of the following. The compound described in claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof. 【Transformation 60】 【change】 【change】

8. Antibody-drug conjugate (ADC), The antibody-drug conjugate is characterized in that it is an antibody-drug conjugate (ADC) formed by coupling a compound of formula A described in claim 1 with an antibody.

9. The composite is as shown in formula B, 【Chemistry 61】 Here, Ab is an antibody, L is a linker, D is a drug, n is characterized by being an integer or decimal number between 1 and 10. The antibody-drug conjugate according to claim 8.

10. The antibody is characterized by comprising antigen-binding fragments, nanobodies, chimeric antibodies, bivalent antibodies, and / or polyvalent antibodies. The antibody-drug conjugate according to claim 8.

11. The antibody or nanobody or its fusion protein is characterized by targeting a target selected from the group consisting of TF, HER2, EGFR, HER3, BCMA, B7-H3, CD73, AXL, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H6, GPC3, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, Trop2, TIGIT, LAG-3, TLR7, or a combination thereof. The antibody-drug conjugate according to claim 8.

12. The antibody is characterized by being an antibody, nanobody, or fusion protein that targets TF. The antibody-drug conjugate according to claim 8.

13. The antigen-binding fragment of the antibody or nanobody targeting the TF nanoparticles is characterized by having CDR1 as shown in SEQ ID NO. 1, CDR2 as shown in SEQ ID NO. 2, and CDR3 as shown in SEQ ID NO.

3. Claim 12: The antibody-drug conjugate.

14. An antibody-drug conjugate, The antibody-drug conjugate comprises the structure shown in formula (B), 【Transformation 62】 Herein, Q is a linker group that can be coupled to an antibody, Z1 is a hydrophilic group containing a hydroxyl group and an amino group-containing glucose group, X is a linking group, P1 is a polypeptide residue, P2 is a directly bound or para-aminobenzoic acid ester (PABC) group, D is an antitumor drug, n is an integer from 1 to 24, Ab is an antibody or a nanobody fusion protein, and m = 1 to 8, characterized in that the antibody-drug conjugate is described above.

15. A pharmaceutical composition, (a) an antibody-drug conjugate according to claim 8 or 14 or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient, comprising the pharmaceutical composition.

16. Uses of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 8 or 14, or a pharmaceutical composition containing the conjugate or a pharmaceutically acceptable salt thereof, in the preparation of an antitumor drug or cancer therapeutic agent.

17. The aforementioned cancer is characterized by being selected from the group consisting of lung cancer, liver cancer, breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, colorectal cancer, gastric cancer, glioma, and mesothelioma. The use described in claim 16.

18. A method for preparing an antibody-drug conjugate according to claim 8 or 14, (1) The step of reacting the antibody with the reducing reagent in a buffer to obtain the reduced antibody, (2) A method for preparing an antibody-drug conjugate according to claim 8 or 14, characterized by comprising the step of crosslinking (coupling) a compound shown in formula A and the reduced antibody obtained in step (1) in a mixture of a buffer and an organic solvent to obtain an antibody-drug conjugate B.

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